Trypsin Exposure Research Articles

Is there a duration-characteristic relationship for trypsin exposure on tendon? A study on anterior cruciate ligament reconstruction in a rabbit model.

Decellularized allograft tendons are highly regarded for their accessibility and the reduced risk of immune rejection, making them a promising choice for grafting due to their favorable characteristics. However, effectively integrating reconstructed tendons with host bone remains a significant clinical challenge. This study aims to investigate the relationship between the duration of tendon exposure to trypsin and its impact on tendon biomechanical properties and healing capacity. Morphological assessments and biochemical quantifications were conducted. Allograft tendons underwent heterotopic transplantation into the anterior cruciate ligament (ACL) in a rabbit model, with specimens harvested 6 weeks post-surgery for a comparative analysis of cell adhesion strength and mechanical performance. Duration-response curves were constructed using maximum stress and cell adhesion quantity as primary indicators. The trypsin treatment enhanced cell adhesion on the tendon surface. Adhesion rates in the control group vs. the experimental groups were as follows: 3.10 ± 0.56% vs. 4.59 ± 1.51%, 5.36 ± 1.24%, 6.12 ± 1.98%, and 8.27 ± 2.34% (F = 6.755, p = 0.001). However, increasing treatment duration led to a decline in mechanical properties, with the ultimate load (N) in the control vs. experimental groups reported as 103.30 ± 10.51 vs. 99.59 ± 4.37, 93.15 ± 12.38, 90.42 ± 7.87, and 82.68 ± 6.89, F = 4.125 (p = 0.013). The findings reveal an increasing trend in adhesion effectiveness with prolonged exposure duration, while mechanical strength declines. The selection of the optimal processing duration should involve careful consideration of the benefits derived from both outcomes.

Does Trypsin Oral Spray (Viruprotect®/ColdZyme®) Protect against COVID-19 and Common Colds or Induce Mutation? Caveats in Medical Device Regulations in the European Union.

Background: nasal or oral sprays are often marketed as medical devices (MDs) in the European Union to prevent common cold (CC), with ColdZyme®/Viruprotect® (trypsin/glycerol) mouth spray claiming to prevent colds and the COVID-19 virus from infecting host cells and to shorten/reduce CC symptoms as an example. We analyzed the published (pre)-clinical evidence. Methods: preclinical: comparison of in vitro tests with validated host cell models to determine viral infectivity. Clinical: efficacy, proportion of users protected against virus (compared with non-users) and safety associated with trypsin/glycerol. Results: preclinical data showed that exogenous trypsin enhances SARS-CoV-2 infectivity and syncytia formation in host models, while culture passages in trypsin presence induce spike protein mutants. The manufacturer claims >98% SARS-CoV-2 deactivation, although clinically irrelevant as based on a tryptic viral digest, inserting trypsin inactivation before host cells exposure. Efficacy and safety were not adequately addressed in clinical studies or leaflets (no COVID-19 data). Protection was obtained among 9–39% of users, comparable to or lower than placebo-treated or non-users. Several potential safety risks (tissue digestion, bronchoconstriction) were identified. Conclusions: the current European MD regulations may result in insufficient exploration of (pre)clinical proof of action. Exogenous trypsin exposure even raises concerns (higher SARS-CoV-2 infectivity, mutations), whereas its clinical protective performance against respiratory viruses as published remains poor and substandard.

Trypsin Induced Degradation of Amyloid Fibrils.

Proteolytic enzymes are known to be involved in the formation and degradation of various monomeric proteins, but the effect of proteases on the ordered protein aggregates, amyloid fibrils, which are considered to be extremely stable, remains poorly understood. In this work we study resistance to proteolytic degradation of lysozyme amyloid fibrils with two different types of morphology and beta-2-microglobulun amyloids. We showed that the proteolytic enzyme of the pancreas, trypsin, induced degradation of amyloid fibrils, and the mechanism of this process was qualitatively the same for all investigated amyloids. At the same time, we found a dependence of efficiency and rate of fibril degradation on the structure of the amyloid-forming protein as well as on the morphology and clustering of amyloid fibrils. It was assumed that the discovered relationship between fibrils structure and the efficiency of their degradation by trypsin can become the basis of a new express method for the analysis of amyloids polymorphism. Unexpectedly lower resistance of both types of lysozyme amyloids to trypsin exposure compared to the native monomeric protein (which is not susceptible to hydrolysis) was attributed to the higher availability of cleavage sites in studied fibrils. Another intriguing result of the work is that the cytotoxicity of amyloids treated with trypsin was not only failing to decline, but even increasing in the case of beta-2-microglobulin fibrils.

An investigation on the correlation between the mechanical property change and the alterations in composition and microstructure of a porcine vascular tissue underwent trypsin-based decellularization treatment.

The nonlinear pseudoelastic behavior of a native/decellularized vascular tissue is closely related to the detailed composition and microstructure of the extracellular matrix and is important in maintaining the patency of a small-caliber vascular graft. A commonly used enzyme-detergent based decellularization protocol is effective in cell component removal but it also changes the microstructure and composition of the decellularized tissues. Previous studies provide limited information to correlate the mechanical property change with the alterations in composition and microstructure in a decellularization process. In this study, the correlations were studied by implementing a previously established fiber-progressive-engagement model to describe the nonlinear pseudoelastic behavior of a vascular tissue and to evaluate the effects of trypsin concentration and exposure duration on porcine coronary artery decellularization RESULTS: Results showed that tissue length and width increased and thickness and wet weight decreased with the exposure of trypsin. The effects of trypsin exposure times on the four mechanical parameters, i.e. initial strain, turning strain, initial modulus and stiffness modulus, in the longitudinal and circumferential directions were similar, but stronger in the circumferential direction. Major components of the extracellular matrix were vulnerable to the trypsin-based decellularization process. The decreases in initial and turning strain and the increase in initial modulus in circumferential direction were correlated with the significant decrease of collagen and glycosaminoglycans in the media layer. Although trypsin-based decellularization achieved cell component removal and preservation of ultimate tensile stress, the microstructure and composition changed with alterations in the pseudoelastic behavior of the porcine coronary artery. Taken together, the current observations suggested less waviness, early engagement, or re-alignment of insoluble collagen fibers in the media layer, which resulted in turning from anisotropic into isotropic uniaxial mechanical property of porcine vascular tissue. Selecting the proper trypsin concentration (< 0.03-0.5%) and duration (< 12 h) of trypsin exposure in combination with other methods will achieve optimal porcine coronary artery decellularization.

Mesenchymal stem cell detachment with trace trypsin is superior to EDTA for in vitro chemotaxis and adhesion assays

Trypsin is frequently used to dissociate mesenchymal stem cells (MSCs) for in vitro adhesion and chemotaxis assays. However, its potential impact on surface receptor degradation is poorly understood. The purpose of this study was to evaluate the effect of trypsin-EDTA exposure versus PBS-EDTA on MSC surface receptor integrity and function. Primary human MSCs were detached with PBS-EDTA alone, or Cell Dissociation Buffer followed by 30 s exposure to 0.05% w/v trypsin-EDTA (trace trypsin method, TT), or 0.25% w/v trypsin exposure for 2 or 5 min. Cells were characterized for surface integrity of β1 integrin (CD29) and PDGF Receptor (PDGF-R), and assessed in vitro for adhesion to atelocollagen-coated surfaces and migration to PDGF-BB. PBS-EDTA detachment fully preserved receptor integrity but routinely detached only half of the adherent cells and led to cell aggregates that failed to adhere evenly across the Transwell migration insert. Both CD29 and PDGF-R were significantly degraded by 0.25% trypsin detachment for 2 or 5 min compared to the TT method or PBS-EDTA (p < 0.05). Cells migrated optimally to PDGF-BB when detached with the TT method (3.1-fold vs α−MEM, p = 0.01). Cells attached optimally to atelocollagen when detached using the TT method or PBS-EDTA (6- to 10-fold vs 0.25% trypsin, p < 0.01). CDB followed by trace trypsin-EDTA exposure is recommended over PBS-EDTA to produce a single-cell MSC suspension that preserves receptor integrity and more reproducible receptor-mediated responses.

Impact of microwave irradiation on enzymatic activity at constant bulk temperature is enzyme-dependent

Microwave heating has been shown to be an effective method in promoting certain chemical reactions. We previously showed that exposure of trypsin from porcine pancreas and its substrate to low power microwave, while the bulk temperature is kept constant, leads to significant increases of the activity of trypsin. We report here that acceleration of enzymatic reactions by microwave at constant bulk temperature is enzyme-dependent. In this regard, the enzymatic activity of α-amylase and alkaline phosphatase towards the hydrolysis of starch and 4-nitrophenyl phosphate is not affected by exposure to microwave (10W), while the reaction temperature is maintained unchanged.

Efficient Subculture Process for Adherent Cells by Selective Collection Using Cultivation Substrate Vibration.

Cell detachment and reseeding are typical operations in cell culturing, often using trypsin exposure and pipetting, even though this process is known to damage the cells. Reducing the number of detachment and reseeding steps might consequently improve the overall quality of the culture, but to date this has not been an option. This study proposes the use of resonant vibration in the cell cultivation substrate to selectively release adherent calf chondrocyte cells: Some were released from the substrate and collected while others were left upon the substrate to grow to confluence as a subculture-without requiring reseeding. An out-of-plane vibration mode with a single nodal circle was used in the custom culture substrate. At a maximum vibration amplitude of 0.6 µm, 84.9% of the cells adhering to the substrate were released after 3 min exposure, leaving a sufficient number of cells for passage and long-term cell culture, with the greatest cell concentration along the nodal circle where the vibration was relatively quiescent. The 72-h proliferation of the unreleased cells was 20% greater in number than cells handled using the traditional method of trypsin-EDTA (0.050%) release, pipette collection, and reseeding. Due to the vibration, it was possible to reduce the trypsin-EDTA used for selective release to only 0.025%, and in doing so the cell number after 72 h of proliferation was 42% greater in number than the traditional technique.

On-demand dissolution of 3D synthetic extracellular matrix for systems biology assays

Event Abstract Back to Event On-demand dissolution of 3D synthetic extracellular matrix for systems biology assays Jorge Valdez1 and Linda Griffith1 1 Massachusetts Institute of Technology, Biological Engineering, United States Introduction: Three-dimensional cell culture formats are desirable in applications ranging from mechanistic analysis of tissue behavior to modeling human responses to new drugs. There is a growing interest in analyzing paracrine interactions between stroma, epithelia, and immune cells in 3D physiological cultures, however the commonly-employed proteolytic methods used to recover cells from 3D gels are often inefficient and can impair fidelity of cytokine measurements and cell surface protein expression, and are not well-suited to allow recovery of locally-released cytokines. Here, we present a method for rapidly (<5 mins) dissolving synthetic polyethylene glycol (PEG)-based matrices using a bacterial transpeptidase with almost no known substrates in native mammalian proteins, in a manner that preserves cell viability and phenotype upon recovery from the gel, and greatly preserves cytokine levels for multiplex measurements compared common proteases. In brief, the approach relies on inclusion of a substrate for the enzyme in the gel crosslinks, allowing the gel to be dissolved by addition of the enzyme and a second simple substrate for the transpeptidase; hence it is translatable to any synthetic gel system. The approach is robust to a range of crosslinking densities, PEG macromer properties, and gel functionalities. Materials and Methods: Endometrial stromal and epithelial cells were encapsulated in PEG gels formed by either norbornene or vinyl sulfone crosslinking with the peptide sequences containing motifs recognized by metalloproteinases in addition to the LPRTG Sortase A (sortA) substrate. PEG macromers were partially functionalized with fluorescein-labelled RGD for dissolution characterization. Epithelial cells were cultured for 5 days before gel dissolution. Gels were dissolved by addition of recombinantly-produced sortA[1],[2] and the sortase substrate GGG. Cell viability upon recovery was measured with trypan blue and viability within gels was evaluated with a live/dead assay using confocal microscopy. Cytokine levels after exposure of sortA, trypsin, or liberase were measured by Luminex according to vendor’s specifications. Results and Discussion: We used sortase chemistry to recover cells from a 3D PEG hydrogel in <5 minutes without decreasing their viability (Fig. 1). We characterized the dissolution method across a wide range of relevant hydrogel mechanical conditions. We observed that sortase mediated-gel degradation preserved gland structures formed by epithelial cells. We compared the effect of sortase/GGG, trypsin, and liberase, on cytokine level measurements by Luminex and saw 16 out of 27 cytokine signals were annihilated by trypsin and/or liberase, while only IL-15 was affected by SrtA (Fig. 2). Conclusions: We developed a robust, easily transferable system for recovering cells from 3D matrices in a bio-orthogonal matter. This method does not affect cell viability, preserves cytokine levels compared to trypsin and collagenase, and could be extremely useful in cell signaling studies and morphogenesis assays. Funding from NSF EBICS center

Abstract 020: A Mitochondrial Renin-Angiotensin System: Internalization of Angiotensinogen

There is compelling evidence for actions of an intracellular renin-angiotensin system (RAS) in various cell organelles including the endoplasmic reticulum, nucleus and the mitochondria (Mito). Indeed, angiotensin (Ang) AT1 and AT2 receptor subtypes were functionally linked to Mito respiration and nitric oxide production, respectively in a previous study. Since elucidation of mitochondrial pathways for expression of RAS protein components as well as Ang II or Ang-(1-7) is equivocal at this time, we undertook a biochemical analysis of the Mito RAS from adult male sheep kidney. Cortical Mito were isolated by differential centrifugation and a discontinuous Percoll gradient. Purified Mito were co-enriched in the voltage-dependent anion channel, an outer Mito membrane marker as well as ATP synthase, an inner membrane marker. Angiotensinogen (Aogen; 55 kDa) was detected in Mito extracts by an Aogen antibody to an internal sequence of the protein, but not with an antibody directed against the Ang I N-terminus. Two different renin antibodies identified a major 35 kDa protein band in the isolated Mito. Using the Ang I-directed Aogen antibody, active renin was confirmed by hydrolysis of Aogen that was abolished by aliskiren; however, trypsin exposure did not increase renin activity in the Mito. A pro-renin receptor (PRR) antibody failed to identify proteins in three Mito preparations, but revealed a prominent band in renal cortical membranes that corresponds to the size of PRR. Angiotensin peptides were quantified by three direct RIAs; the Mito content of Ang II and Ang-(1-7) were higher as compared to Ang I [23 ± 8 and 58 ± 17 vs. 2 ± 1 fmol/mg protein; p&lt;0.01, n=3]. Additionally, both neprilysin and thimet oligopeptidase activities that processed Ang I to Ang-(1-7) were evident. Finally, cortical Mito internalized radiolabeled Aogen at a rate of 33 ± 9 fmol/min/mg protein (n=3) at 37°C. The subsequent analysis of the labeled Mito by SDS-gel fractionation revealed a predominant radioactive band of 55 kDa for Aogen. Collectively, our data suggest that the internalization of Aogen and subsequent processing by active renin may yield des-[Ang I]-Aogen and the active peptides Ang II and Ang-(1-7) that may potentially contribute to mitochondrial function within the kidney.

Protease-Activated Receptor-2 Up-Regulates Transient Receptor Potential Vanilloid 4 Function in Mouse Esophageal Keratinocyte.

The reflux of pancreatic-duodenal fluids is implicated in the pathophysiology of proton-pump inhibitor-resistant gastroesophageal reflux disease (GERD). Protease-activated receptor-2 (PAR-2) is activated by proteases, the pancreatic enzyme, trypsin, and the activated PAR-2 enhances transient receptor potential vanilloid 4 (TRPV4) function in neurons. TRPV4 stimulates ATP exocytosis in conjunction with the vesicular nucleotide transporter, which mediates mechano-transduction and vagal stimulation. The aim of the present study was to verify whether the activated PAR-2 up-regulates TRPV4 function in mouse esophageal keratinocytes, which may link to the pathophysiology in PPI-resistant GERD. TRPV4 and PAR-2 expressions were detected by RT-PCR, immunostaining, and western blotting in mouse esophageal keratinocytes. The functional response of TRPV4 to esophageal keratinocytes was analyzed using a Ca(2+) imaging system. Cellular ATP release was examined by luciferase-luciferin reaction. TRPV4 phosphorylation was studied by immunoprecipitation and western blotting. PAR-2 and TRPV4 mRNAs and proteins were expressed in esophageal keratinocytes. Pre-treatment with trypsin significantly increased the responses to TRPV4 activator in esophageal keratinocytes, probably via the phosphorylation of serine residue of TRPV4 by protein kinase C and resulted in cellular ATP release from the cells. Activated PAR-2 with trypsin exposure up-regulated TRPV4 function and increased ATP release in mouse esophageal keratinocytes. This mechanism might be related to the pathophysiology of GERD, especially non-erosive GERD.

Independent Pathways Can Transduce the Life-Cycle Differentiation Signal in Trypanosoma brucei

African trypanosomes cause disease in humans and livestock, generating significant health and welfare problems throughout sub-Saharan Africa. When ingested in a tsetse fly bloodmeal, trypanosomes must detect their new environment and initiate the developmental responses that ensure transmission. The best-established environmental signal is citrate/cis aconitate (CCA), this being transmitted through a protein phosphorylation cascade involving two phosphatases: one that inhibits differentiation (TbPTP1) and one that activates differentiation (TbPIP39). Other cues have been also proposed (mild acid, trypsin exposure, glucose depletion) but their physiological relevance and relationship to TbPTP1/TbPIP39 signalling is unknown. Here we demonstrate that mild acid and CCA operate through TbPIP39 phosphorylation, whereas trypsin attack of the parasite surface uses an alternative pathway that is dispensable in tsetse flies. Surprisingly, glucose depletion is not an important signal. Mechanistic analysis through biophysical methods suggests that citrate promotes differentiation by causing TbPTP1 and TbPIP39 to interact.

Effect of disaccharides on the stabilization of bovine trypsin against detergent and autolysis

Osmolytes have been reported to stabilize biomolecules and even whole organisms against exposure to adverse environmental conditions. In this work, we report for the first time the use of some of these osmolytes, viz., the disaccharides trehalose and sucrose, in the stabilization of bovine trypsin against exposure to the anionic detergent sodium dodecyl sulfate and autolysis. Exposure of trypsin to SDS at a molar ratio of 1:45 led to decrease in trypsin activity by 61%. In the presence of 1 M sucrose and 1 M trehalose, the residual trypsin activity was found to increase to that of original enzyme activity. These two disaccharides were also found to slow down the rate of autolysis, resulting in residual activities of 80 and 88%, respectively, after incubation for 24 h. Active site titration showed retention of the fraction of active sites in the presence of trehalose. Fluorescence and CD spectroscopies were used to decipher the probable mechanism of this protective role of the disaccharides. Although complete resumption of secondary structure was not seen in the presence of the two disaccharides, the spectra of trypsin in the presence of stabilizers resembled the spectrum of native trypsin and were significantly different from the spectrum of detergent-denatured enzyme. Correlating the data obtained from spectroscopy with those obtained from activity assay, we propose that the retention of secondary structure of the enzyme is largely responsible for the retention of the functionally active form of trypsin.

Optimization of Ca v1.2 screening with an automated planar patch clamp platform

Introduction: Ca v1.2 channels play an important role in shaping the cardiac action potential. Screening pharmaceutical compounds for Ca v1.2 block is very important in developing drugs without cardiac liability. Ca v1.2 screening has been traditionally done using fluorescence assays, but these assays have some limitations. Patch clamping is considered the gold standard for ion channel studies, but is very labor intensive. The purpose of this study was to develop a robust medium throughput Ca v1.2 screening assay in PatchXpress ® 7000A by optimizing cell isolation conditions, recording solutions and experimental parameters. Under the conditions established, structurally different standard Ca v1.2 antagonists and an agonist were tested. Methods: HEK-293 cells stably transfected with hCa v1.2 L-type Ca channel were used. For experiments, cells were isolated using 0.05% Trypsin. Currents were recorded in the presence of 30 mM extracellular Ba 2+ and low magnesium intracellular recording solution to minimize rundown. Ca v1.2 currents were elicited from a holding potential of − 60 mV at 0.05 Hz to increase pharmacological sensitivity and minimize rundown. Test compounds were applied at increasing concentrations for 5 min followed by a brief washout. Results: Averaged peak Ca v1.2 current amplitudes were increased from 10 pA/pF to 15 pA/pF by shortening cell incubation and trypsin exposure time from 2.5 min at 37 °C to 1 min at room temperature and adding 0.2 mM cAMP to the intracellular solution. Rundown was minimized from 2%/min to 0.5%/min by reducing the intracellular free Mg 2+ from 2.7 mM to 0.2 mM and adding 100 nM Ca 2+. Under the established conditions, we tested 8 structurally different antagonists and an agonist. The IC 50 values obtained ranked well against published values and results obtained using traditional clamp experiments performed in parallel using the expressed cell line and native myocytes. Discussion: This assay can be used as a reliable pharmacological screening tool for Ca v1.2 block to assess compounds for cardiac liability during lead optimization.

130. Trypsin Stimulates Mitogen Activated Protein Kinases and Cell Growth in Barrett’S Esophagus

Background: Barrett’s esophagus (BE) is associated with significant reflux of Gastro-duodenal contents. In addition to containing bile salts, duodenal juice is also rich in proteases including trypsin. Protease activated receptors (PAR) are a family of transmembrane receptors activated by proteolytic cleavage which are capable of activating pro-inflammatory and growth signaling in a variety of cell types. We hypothesized that trypsin is capable of activating growth signaling and cell growth in Barrett’s metaplasia. Methods: The Human non-neoplastic telomerase immortalized cell line BAR-T was used as a model of Barrett’s esophagus. Erk and p38 MAPK activation was measured by Western blotting for phosphorylated and total proteins. Cell growth was measured by Coulter counter direct cell counts 24 hours after agonist exposure. Statistical significance was determined by ANOVA. Results: A five minute exposure to trypsin produced both dose and time dependent activation of both Erk and p38 MAPK in Bar-T cells (Figure 1). Maximal Erk and p38 activation occurred within 10 minutes of trypsin exposure.

Nucleotidase Activity of CD39/NTPDase1 Is Dependent on Internal Proteolytic Cleavage Which Contributes to Lipid Raft Localization.

Brief trypsin exposure increases apyrase activity in hCD39 expressing cells, as previously reported (Schulte am Esch et al, Biochemistry 38:2248, 1999). Since regulated proteolytic cleavage of CD39 would allow for a rapid response to extracellular stimuli, we studied the relationship between observed CD39 cleavage and enzymatic activity. We generated N- and C-terminal VP16-tagged hCD39 to study CD39 expression, processing, and activity in transiently transfected HEK 293 cells. We found that optimal enzymatic activity of CD39 indeed depends on incorporation into cholesterol-rich plasma membrane domains (lipid "rafts"). Membrane fractions from hCD39 -transfected 293 cells readily hydrolyze ATP. Pretreatment of 293 cells with the cholesterol-depleting agent methyl β cyclodextrin (MBCD) results in a dose-dependent decrease in ATPase activity. In addition, treatment of isolated membranes with MBCD also decreases enzymatic activity. We next performed Western blot analyses of membranes prepared from hCD39-transfected 293 cells treated with membrane-impermeant crosslinking agents. These experiments demonstrated a dose-dependent, MBCD-reversible decrease in monomeric CD39. Taken together, these data demonstrate that CD39 enzyme activity resides in raft-localized CD39. Western blots of membrane fractions from cells transfected with N- or C-terminal VP16-tagged hCD39 show partial cleavage of full-length CD39 to yield a 20kDa N-terminal and 50 kDa C-terminal fragments. Biotinylation studies established that both fragments are expressed on the cell surface. As with full-length CD39, crosslinking results in dose-dependent decreases of both monomeric species. Moreover, prior cholesterol depletion with MBCD abolishes crosslinking. Since the cleavage products of full-length CD39 are expressed on the cell surface and localize to lipid rafts, we examined the relation between CD39 cleavage, ATPase activity and lipid raft localization using a panel of cell permeable protease inhibitors. 293 cells transfected with N-terminal VP16-tagged CD39 were treated with AEBSF (serine protease inhibitor), zYVAD.fmk (caspase inhibitor), zLLY.fmk (calpain inhibitor) or the furin inhibitor Furin I. All inhibitors resulted in dose-dependent decreases in formation of the VP16-tagged N-terminal fragment. Concomitantly, ATPase assays of the membrane fractions demonstrated a corresponding dose-dependent decrease in enzymatic activity. Finally, we established that CD39 cleavage promotes raft localization, since protease inhibition decreased the fraction of CD39 susceptible to crosslinking with all inhibitors tested. In summary, we have established that generation of optimally active, raft-localized CD39 requires prior limited proteolysis of the full-length molecule. Activation of caspase-1 by exposure of cells to ATP leads to processing and release of interleukin family members. We propose that purinergic signaling might also enhance CD39 cleavage in vascular cells by an as yet unidentified protease. Our data suggest that subsequent increased cell surface apyrase activity leads to dampening of purinergic signaling and a resulting increase in antithrombotic activity. Of note, we identified an alternately spliced isoform of CD39 which inhibits cleavage of the full-length molecule.

Exploring the protease mediated conformational stability in a trypsin inhibitor from Archidendron ellipticum seeds

A Kunitz proteinase inhibitor from Archidendron ellipticum seeds (AeTI) was purified and complexed with bovine trypsin and chymotrypsin. The stoichiometric stability of AeTI with its interacting proteinases was then investigated using spectrophotometric, size exclusion chromatography (HPLC system), Western blotting and circular dichroism (CD) studies. All the methods were remarkably similar in revealing the preference of trypsin over chymotrypsin by AeTI for complex formation. Both Western blotting as well as spectrophotometry based assays for competition experiments indicated that trypsin displaces chymotrypsin from a previously formed AeTI–chymotrypsin complex. Chemical modification of lysine and arginine by TNBS and CHD treatments, respectively, suggested a lysine as the active site residue and also indicated the presence of a single protease-binding site for AeTI. CD of native AeTI showed a sharp minimum at 200 nm and deconvolution of the CD spectra revealed it to be an unordered protein possessing high β-sheet content. Complex formation of AeTI with trypsin induces a fractional switchover of its unordered structure towards the β-sheet fraction but lacked any such conversion in the presence of chymotrypsin. Prolonged exposure of excess trypsin generates conformational modifications both in the secondary and the tertiary structures.

Trypsin-Sensitive Modulation of Intestinal Epithelial MD-2 as Mechanism of Lipopolysaccharide Tolerance

Intestinal epithelial cells (IEC) are constantly exposed to both high concentrations of the bacterial ligand LPS and the serine protease trypsin. MD-2, which contains multiple trypsin cleavage sites, is an essential accessory glycoprotein required for LPS recognition and signaling through TLR4. The aim of this study was to characterize the expression and subcellular distribution of intestinal epithelial MD-2 and to delineate potential functional interactions with trypsin and then alteration in inflammatory bowel disease (IBD). Although MD-2 protein expression was minimal in primary IEC of normal colonic or ileal mucosa, expression was significantly increased in IEC from patients with active IBD colitis, but not in ileal areas from patients with severe Crohn's disease. Endogenous MD-2 was predominantly retained in the calnexin-calreticulin cycle of the endoplasmic reticulum; only a small fraction was exported to the Golgi. MD-2 expression correlated inversely with trypsin activity. Biochemical evidence and in vitro experiments demonstrated that trypsin exposure resulted in extensive proteolysis of endogenous and soluble MD-2 protein, but not of TLR4 in IEC, and was associated with desensitization of IEC to LPS. In conclusion, the present study suggests that endoplasmic reticulum-associated MD-2 expression in IBD may be altered by ileal protease in inflammation, leading to impaired LPS recognition and hyporesponsiveness through MD-2 proteolysis in IEC, thus implying a physiologic mechanism that helps maintain LPS tolerance in the intestine.

Renin, Prorenin and the Putative (Pro)renin Receptor

Renin is an aspartic protease that consists of 2 homologous lobes. The cleft in between contains the active site with 2 catalytic aspartic residues.1 Unlike other aspartic proteases such as pepsin or cathepsin D, renin is monospecific and only cleaves angiotensinogen, to generate angiotensin (Ang) I. Ang I is the precursor of the active end-product of the renin-angiotensin system (RAS), Ang II. Renin has also been called active renin to underline that an enzymatically inactive form of renin exists. In 1971, Lumbers found that amniotic fluid, left at low pH in the cold, acquired renin activity.2 Later, Skinner described a similar phenomenon in plasma.3 Acidification was not strictly necessary for this increase in Ang I-generating activity, because incubation at low temperature also increased renin activity, albeit to only 15% of activity after acidification. Soon it was postulated that this inactive, but activatable “big” renin (its molecular weight was 5 kDa higher than that of renin) was the biosynthetic precursor of renin. Hence, it was named prorenin. Only with the cloning of the renin gene in 1984 was prorenin definitively proved to be the precursor of renin.4 For reasons that are unknown, prorenin circulates in human plasma in excess to renin, sometimes at concentrations that are 100-times higher.5 Prorenin has also been demonstrated in plasma of cat, dog, cattle, pig, horse, sheep, rabbit, rat, and mouse. A 43-amino acid N-terminal propeptide explains the absence of enzymatic activity of prorenin. This propeptide covers the enzymatic cleft and obstructs access of angiotensinogen to the active site of renin. (Pro)renin is synthesized as a preprohormone. It contains a signal peptide that directs the protein to the endoplasmic reticulum and ultimately to the exterior of the cell. Both renin and prorenin can be fractionated into multiple species by isoelectric focusing. …

Targeted Modification of Atrial Electrophysiology by Homogeneous Transmural Atrial Gene Transfer

Safe and effective myocardial gene transfer remains elusive. Heterogeneous ventricular gene delivery has been achieved in small mammals but generally with methods not readily transferable to the clinic. Atrium-specific gene transfer has not yet been reported. We hypothesized that homogeneous atrial gene transfer could be achieved by direct application of adenoviral vectors to the epicardial surface, use of poloxamer gel to increase virus contact time, and mild trypsinization to increase virus penetration. We "painted" recombinant adenovirus encoding the reporter gene Escherichia coli beta-galactosidase directly onto porcine atria. Investigational variables included poloxamer use, trypsin concentration, and safety. Using the painting method, we modified the atrial phenotype with an adenovirus expressing HERG-G628S, a long-QT-syndrome mutant. Our results showed that application of virus with poloxamer alone resulted in diffuse epicardial gene transfer with negligible penetration into the myocardium. Dilute trypsin concentrations allowed complete transmural gene transfer. After trypsin exposure, echocardiographic left atrial diameter did not change. Left atrial function decreased on postoperative day 3 but returned to baseline by day 7. Tissue tensile strength was affected only in the 1% trypsin group. HERG-G628S gene transfer prolonged atrial action potential duration and refractory period without affecting ventricular electrophysiology. We show complete transmural atrial gene transfer by this novel painting method. Adaptation of the method could allow application to other tissue targets. Use with functional proteins in the atria could cure or even prevent diseases such as atrial fibrillation or sinus node dysfunction.

Neutrophil elastase activates near-silent epithelial Na+channels and increases airway epithelial Na+transport

Neutrophil elastase is a serine protease that is abundant in the airways of individuals with cystic fibrosis (CF), a genetic disease manifested by excessive airway Na(+) absorption and consequent depletion of the airway surface liquid layer. Although endogenous epithelium-derived serine proteases regulate epithelial Na(+) transport, the effects of neutrophil elastase on epithelial Na(+) transport and epithelial Na(+) channel (ENaC) activity are unknown. Low micromolar concentrations of human neutrophil elastase (hNE) applied to the apical surface of a human bronchial cell line (16HBE14o-/beta gamma) increased Na(+) transport about twofold. Similar effects were observed with trypsin, also a serine protease. Proteolytic inhibitors of hNE or trypsin selectively abolished the enzyme-induced increase of epithelial Na(+) transport. At the level of the single channel, submicromolar concentrations of hNE increased activity of near-silent ENaC approximately 108-fold in patches from NIH-3T3 cells expressing rat alpha-, beta-, and gamma-ENaC subunits. However, no enzyme effects were observed on basally active ENaCs. Trypsin exposure following hNE revealed no additional increase in amiloride-sensitive short-circuit current or in ENaC activity, suggesting these enzymes share a common mode of action for increasing Na(+) transport, likely through proteolytic activation of ENaC. The hNE-induced increase of near-silent ENaC activity in CF airways could contribute to Na(+) hyperabsorption, reduced airway surface liquid height, and dehydrated mucus culminating in inefficient mucociliary clearance.