Cryo-EM structures reveal the activation and substrate recognition mechanism of human enteropeptidase.

Enteropeptidase (EP) initiates intestinal digestion by proteolytically processing trypsinogen, generating catalytically active trypsin. EP dysfunction causes a series of pancreatic diseases including acute necrotizing pancreatitis. However, the molecular mechanisms of EP activation and substrate recognition remain elusive, due to the lack of structural information on the EP heavy chain. Here, we report cryo-EM structures of human EP in inactive, active, and substrate-bound states at resolutions from 2.7 to 4.9 Å. The EP heavy chain was observed to clamp the light chain with CUB2 domain for substrate recognition. The EP light chain N-terminus induced a rearrangement of surface-loops from inactive to active conformations, resulting in activated EP. The heavy chain then served as a hinge for light-chain conformational changes to recruit and subsequently cleave substrate. Our study provides structural insights into rearrangements of EP surface-loops and heavy chain dynamics in the EP catalytic cycle, advancing our understanding of EP-associated pancreatitis.

Comparison of Periplasmic and Cytoplasmic Expression of Bovine Enterokinase Light Chain in E. coli.

Enterokinase enzyme is widely used in production of recombinant proteins. This enzyme is isolated from the intestine and recognizes a specific cleavage site (X↓LYS-ASP4). Several studies have been performed to produce recombinant active enterokinase. In this study, the coding sequence of bovine enteropeptidase light chain (bEKL) was isolated from Iranian Sarabi cattle and its expression was investigated in the periplasm and cytoplasm of E. coli by two different expression vectors, pET22 and pET32RH. RNA was extracted from the duodenum part of cattle, cDNA was amplified, the enterokinase light chain coding fragment was cloned and the expression was examined by SDS-PAGE analysis. The higher amounts of soluble enterokinase as a fusion with thioredoxin (Trx) were detected in cytoplasmic expression. The functional enterokinase was purified with a yield of 45 mg per litter by two-steps Ni2+ affinity chromatography. The effective activity of the enzyme implies that it can be produced in large scale for biotechnological applications.

Targeting Enteropeptidase with Reversible Covalent Inhibitors To Achieve Metabolic Benefits.

Inhibition of the serine protease enteropeptidase (EP) opens a new avenue to the discovery of chemotherapeutics for the treatment of metabolic diseases. Camostat has been used clinically for treating chronic pancreatitis in Japan; however, the mechanistic basis of the observed clinical efficacy has not been fully elucidated. We demonstrate that camostat is a potent reversible covalent inhibitor of EP, with an inhibition potency (k inact/KI) of 1.5 × 104 M-1s-1 High-resolution liquid chromatography-mass spectrometry (LC-MS) showed addition of 161.6 Da to EP after the reaction with camostat, consistent with insertion of the carboxyphenylguanidine moiety of camostat. Covalent inhibition of EP by camostat is reversible, with an enzyme reactivation half-life of 14.3 hours. Formation of a covalent adduct was further supported by a crystal structure resolved to 2.19 Å, showing modification of the catalytic serine of EP by a close analog of camostat, leading to formation of the carboxyphenylguanidine acyl enzyme identical to that expected for the reaction with camostat. Of particular note, minor structural modifications of camostat led to changes in the mechanism of inhibition. We observed from other studies that sustained inhibition of EP is required to effect a reduction in cumulative food intake and body weight, with concomitant improved blood glucose levels in obese and diabetic leptin-deficient mice. Thus, the structure-activity relationship needs to be driven by not only the inhibition potency but also the mechanistic and kinetic characterization. Our findings support EP as a target for the treatment of metabolic diseases and demonstrate that reversible covalent EP inhibitors show clinically relevant efficacy. SIGNIFICANCE STATEMENT: Interest in targeted covalent drugs has expanded in recent years, particularly so for kinase targets, but also more broadly. This study demonstrates that reversible covalent inhibition of the serine protease enteropeptidase is a therapeutically viable approach to the treatment of metabolic diseases and that mechanistic details of inhibition are relevant to clinical efficacy. Our mechanistic and kinetic studies outline a framework for detailed inhibitor characterization that is proving essential in guiding discovery efforts in this area.

Site-specific, covalent immobilization of an engineered enterokinase onto magnetic nanoparticles through transglutaminase-catalyzed bioconjugation.

Enterokinase (EK) is one of the most popular enzymes for the in vitro cleavage of fusion proteins due to its high degree of specificity for the amino-acid sequence (Asp)4-Lys. Enzyme reusability is desirable for reducing operating costs and facilitating the industrial application of EK. In this work, we report the controlled, site-specific and covalent cross-linking of an engineered EKLC on amine-modified magnetic nanoparticles (NH2-MNPs) via microbial transglutaminase-catalyzed bioconjugation for the development of the oriented-immobilized enzyme, namely, EKLC@NH2-MNP biocatalyst. Upon the site-specific immobilization, approximately 90% EKLC enzymatic activity was retained, and the biocatalyst exhibited more than 85% of initial enzymatic activity regardless of storage or reusable stability over a month. The EKLC@NH2-MNP biocatalyst was further applied to remove the His tag-(Asp)4-Lys fusion partner from the His tag-(Asp)4-Lys-(GLP-1)3 substrate fusion protein, result suggested the EKLC@NH2-MNP possessed remarkable reusability, without a significant decrease of enzymatic activity over 10 cycles (P > 0.05). Supported by the unique properties of MNPs, the proposed EKLC@NH2-MNP biocatalyst is expected to promote the economical utilization of enterokinase in fusion protein cleavage.

Effects of N-glycosylation on the biochemical properties of recombinant bEKL expressed in Pichia pastoris.

Enterokinase is an ideal tool protease for cleaving fusion proteins in genetic engineering. The bovine enterokinase light chain (bEKL) produced in Pichia pastoris was shown to be a glycoprotein. To study the effects of N-glycosylation on the biochemical properties of bEKL, the enzyme was deglycosylated via site-directed mutagenesis. The results showed that elimination of the N-glycosylation sites of bEKL (N64, N103 and N165) did not significantly affect the protein secretion level in P. pastoris, but it does greatly influence its enzymatic activity. The N64Q increased the specific activity of the enzyme for GD4K-ß-naphthylamide and improved its catalytic efficiency. Moreover, the glycosylated bEKL is more thermostable than its deglycosylated counterparts. Structural analysis of glycosylated and deglycosylated bEKL revealed that the removal of N-glycosylation did not have pronounced changes on the secondary structure but there was a significant difference in the tertiary structure. In conclusion, this study demonstrated that the effects of glycosylation at different degrees and sites in bEKL were diverse. Moreover, this work will provide theoretical support for designing enzymes on the basis of N-glycosylation to meet the demands of the biochemical industry.

Recombinant production of the insecticidal scorpion toxin BjαIT in Escherichia coli.

Scorpion long-chain insect neurotoxins have important potential application value in agricultural pest control. The difficulty of obtaining natural toxins is the major obstacle preventing analyses of their insecticidal activity against more agricultural insect pests. Here we cloned the insect neurotoxin BjαIT gene into the pET32 expression vector and expressed the resulting thioredoxin (Trx)-BjαIT fusion protein in Escherichia coli. Soluble Trx-BjαIT was expressed at a high level when induced at 18 °C with 0.1 mM isopropyl ß-d-1-thiogalactopyranoside, and it was purified by Ni2+-nitriloacetic acid affinity chromatography. After cleaving the Trx tag with recombinant enterokinase, the digestion products were purified by CM Sepharose FF ion-exchange chromatography, and 1.5 mg of purified recombinant BjαIT (rBjαIT) was obtained from 100 ml of induced bacterial cells. Injecting rBjαIT induced obvious neurotoxic symptoms and led to death in locust (Locusta migratoria) larvae. Dietary toxicity was not observed in locusts. The results demonstrate that active rBjαIT could be obtained efficiently from an E. coli expression system, which is helpful for determining its insecticidal activity against agricultural insect pests.

Preparation of umami octopeptide with recombined Escherichia coli: Feasibility and challenges.

The taste of umami peptide H-Lys-Gly-Asp-Glu-Glu-Ser-Leu-Ala-OH (LGAGGSLA) is controversial. One possible reason for this controversy is the use of chemically synthesized LGAGGSLA to confirm its taste. To explore other ways to further confirm the flavor of LGAGGSLA, we developed a new strategy to prepare a bio-source peptide by adopting a gene engineering method to express LGAGGSLA in recombinant Escherichia coli. In our previous work, we structured the LGAGGSLA recombinant expression system and optimized the culturing conditions for preparing a fusion protein. However, the fusion protein was not cleaved by enterokinase to obtain LGAGGSLA. Because the cleavage conditions of commercial enterokinase were not specific and recombinant engineered bacteria had the potential to be used in industrial processes, in this addendum, we calculated the mass and volume yields of key processing steps in the preparation of LGAGGSLA, and established a model of cleavage conditions with the cleavage ratio of LGAGGSLA. When the LGAGGSLA was confirmed to show umami taste, it is considered as a new umami or umami enhancer. The gene information of LGAGGSLA should have a great potential in the development of new flavor product and food product containing high umami flavor.

Fine-tuning sortase-mediated immobilization of protein layers on surfaces using sequential deprotection and coupling.

Increasing interest in protein immobilization on surfaces has heightened the need for techniques enabling layer-by-layer protein attachment. Here, we report a technique for controlling enzyme-mediated immobilization of layers of protein on the surface using a genetically encoded protecting group. An enterokinase-cleavable peptide sequence was inserted at the N-terminus of bifunctional fluorescent proteins containing Sortase A substrate recognition tags at both ends to control Sortase A-mediated protein immobilization on the surface layer-by-layer. Efficient, sequential immobilization of a second layer of protein using Sortase A required removal of the N-terminal protecting group, suggesting the method enables multilayer synthesis using cyclic deprotection and coupling steps. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:824-831, 2017.

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells.

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

Expression and purification of short hydrophobic elastin-like polypeptides with maltose-binding protein as a solubility tag.

Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. The recombinant expression of hydrophobic elastin-like polypeptides is often difficult because they possess low transition temperatures, and therefore form aggregates at sub-ambient temperatures. To circumvent this difficulty, we expressed in Escherichia coli three hydrophobic ELPs (VPGIG)n with variable lengths (n=20, 40, and 60) in fusion with the maltose-binding protein (MBP). Fusion proteins were soluble and yields of purified MBP-ELP ranged between 66 and 127mg/L culture. After digestion of the fusion proteins by enterokinase, the ELP moiety was purified by using inverse transition cycling. The purified fraction containing ELP40 was slightly contaminated by traces of undigested fusion protein. Purification of ELP60 was impaired because of co-purification of the MBP tag during inverse transition cycling. ELP20 was successfully purified to homogeneity, as assessed by gel electrophoresis and mass spectrometry analyses. The transition temperature of ELP20 was measured at 15.4°C in low salt buffer. In conclusion, this method can be used to produce hydrophobic ELP of low molecular mass.

Improvement of an antibody-enzyme coupling yield by enzyme surface supercharging.

BACKGROUND: Protein cross-coupling reactions demand high yields, especially if the products are intended for bioanalytics, like enzyme-linked immunosorbent assays. Amongst other factors, the coupling yield depends on the concentration of the proteins being used for coupling. Protein supercharging of enzymes can increase the solubility dramatically, which could promote enzyme-antibody coupling reactions. A highly soluble, supercharged variant of the enzyme human enteropeptidase light chain was created by a site-directed mutagenesis of surface amino acids, used for the production of an antibody-enzyme conjugate and compared to the wild type enzyme. RESULTS: Wild type and mutant enzyme could successfully be cross-coupled to an antibody to give antibody-enzyme conjugates suitable for ELISA. Their assay performances and the analysis of the enzyme activities in solution demonstrate that the supercharged version could be coupled to a higher extent, which resulted in better assay sensitivities. The generated conjugate, based on the supercharged enzyme, was feasible as a reporter molecule in a sandwich ELISA and allowed the detection of epidermal growth factor with a detection limit of 15.63 pg (25 pmol/L). CONCLUSION: The highly soluble, surface supercharged, human enteropeptidase light chain mutant provided better yields in coupling the enzyme to an antibody than the wild type. This is most likely related to the higher protein concentration during the coupling. The data suggest that supercharging can be applied favourably to other proteins which have to be covalently linked to other polymers or surfaces with high yields without losses in enzyme activity or specificity.

Do-it-yourself histidine-tagged bovine enterokinase: a handy member of the protein engineer's toolbox.

Enterokinase, a two-chain duodenal serine protease, activates trypsinogen by removing its N-terminal propeptide. Due to a clean cut after the non-primed site recognition sequence, the enterokinase light chain is frequently employed in biotechnology to separate N-terminal affinity tags from target proteins with authentic N-termini. In order to obtain large quantities of this protease, we adapted an in vitro folding protocol for a pentahistidine-tagged triple mutant of the bovine enterokinase light chain. The purified, highly active enzyme successfully processed recombinant target proteins, while the pentahistidine-tag facilitated post-cleavage removal. Hence, we conclude that producing enterokinase in one's own laboratory is an efficient alternative to the commercial enzyme.

Heterogeneous catalysis on the phage surface: Display of active human enteropeptidase.

Enteropeptidase (EC 3.4.21.9) plays a key role in mammalian digestion as the enzyme that physiologically activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the recognition sequence D4K. The high specificity of enteropeptidase makes it a powerful tool in modern biotechnology. Here we describe the application of phage display technology to express active human enteropeptidase catalytic subunits (L-HEP) on M13 filamentous bacteriophage. The L-HEP/C122S gene was cloned in the g3p-based phagemid vector pHEN2m upstream of the sequence encoding the phage g3p protein and downstream of the signal peptide-encoding sequence. Heterogeneous catalysis of the synthetic peptide substrate (GDDDDK-ß-naphthylamide) cleavage by phage-bound L-HEP was shown to have kinetic parameters similar to those of soluble enzyme, with the respective Km values of 19 µM and 20 µM and kcat of 115 and 92 s(-1). Fusion proteins containing a D4K cleavage site were cleaved with phage-bound L-HEP/C122S as well as by soluble L-HEP/C122S, and proteolysis was inhibited by soybean trypsin inhibitor. Rapid large-scale phage production, one-step purification of phage-bound L-HEP, and easy removal of enzyme activity from reaction samples by PEG precipitation make our approach suitable for the efficient removal of various tag sequences fused to the target proteins. The functional phage display technology developed in this study can be instrumental in constructing libraries of mutants to analyze the effect of structural changes on the activity and specificity of the enzyme or generate its desired variants for biotechnological applications.

Human enteropeptidase light chain: bioengineering of recombinants and kinetic investigations of structure and function.

The serine protease enteropeptidase exhibits a high level of substrate specificity for the cleavage sequence DDDDK∼ X, making this enzyme a useful tool for the separation of recombinant protein fusion domains. In an effort to improve the utility of enteropeptidase for processing fusion proteins and to better understand its structure and function, two substitution variants of human enteropeptidase, designated R96Q and Y174R, were created and produced as active (>92%) enzymes secreted by Pichia pastoris with yields in excess of 1.7 mg/Liter. The Y174R variant showed improved specificities for substrates containing the sequences DDDDK (kcat /KM = 6.83 × 106 M⁻¹ sec⁻¹) and DDDDR (kcat /KM = 1.89 × 107 M⁻¹ sec⁻¹) relative to all other enteropeptidase variants reported to date. BPTI inhibition of Y174R was significantly decreased. Kinetic data demonstrate the important contribution of the positively charged residue 96 to extended substrate specificity in human enteropeptidase. Modeling shows the importance of the charge-charge interactions in the extended substrate binding pocket.

Preparation and functional evaluation of RGD-modified streptavidin targeting to integrin-expressing melanoma cells.

The vertical growth stage is the most dangerous stage of melanoma and is often associated with a poor prognosis. The increased invasiveness and metastasis that is typical for vertically growing melanoma are mediated by the molecules of cell adhesion (particularly, integrins). Integrin αvß3, which is abundantly expressed on melanoma cells with high metastatic potentials and is characterized by low expression levels in normal melanocytes, is potentially an attractive target for melanoma diagnostics and therapy. Integrin αvß3 is known to recognize the arginine-glycine-aspartic (RGD) sequence, which has been found in a wide variety of its natural ligands. Here expression vectors bearing the genes of fusion proteins have been constructed for producing these proteins in Escherichia coli. Such fusion proteins consist of a peptidic 'address,' targeting the integrins on melanoma cells, linked to an 'adaptor' for the attachment of a diagnostic or toxic agent. The peptidic 'address' contains the RGD motif, which is stabilized by a disulfide bond to achieve the optimal receptor binding conformation. The 'adaptor' is a tetrameric protein, namely, streptavidin, that is able to achieve high-affinity binding of d-biotin (K(d) = 10(-15) M) and confer avidity to the address peptide. This binding ability facilitates the generation of anti-melanoma diagnostic and therapeutic agents using the appropriate biotin derivatives. These recombinant proteins were purified from the periplasm of E.coli using columns with 2-iminobiotin agarose and demonstrated an ability to adhere to the surface of murine and human melanoma cells.

Fusion expression and purification of four disulfide-rich peptides reveals enterokinase secondary cleavage sites in animal toxins.

Animal toxins are powerful tools for testing the pharmacological, physiological, and structural characteristics of ion channels, proteases, and other receptors. However, most animal toxins are disulfide-rich peptides that are difficult to produce functionally. Here, a glutathione S-transferase (GST) fusion expression strategy was used to produce four recombinant animal toxin peptides, ChTX, StKTx23, BmP01, and ImKTx1, with different isoelectric points from 4.7 to 9.2. GST tags were removed by enterokinase, a widely used and effective commercial protease that cleaves after lysine at the cleavage site DDDDK. Using this strategy, two disulfide-rich animal toxins ChTX and StKTx23 were obtained successfully with a yield of approximately 1-2 mg/l culture. Electrophysiological experiments further showed that these two recombinant toxins showed good bioactivities, indicating that our method was effective in producing large amounts of functional disulfide-rich animal toxins. Interestingly, by analyzing the separated fractions of BmP01, StKTx23, and ImKTx1 using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, four new enterokinase secondary cleavage sites were found, consisting of the sequences "WEYR," "EDK," "QNAR," and "DNDK." To our knowledge, this is the first report of the presence of secondary cleavage sites for commercial enterokinase in animal toxins. These findings will help us use commercial enterokinase appropriately as a cleavage tool in the production of animal toxins.

Dissecting structural basis of the unique substrate selectivity of human enteropeptidase catalytic subunit.

Enteropeptidase is a key enzyme in the digestion system of higher animals. It initiates enzymatic cascade cleaving trypsinogen activation peptide after a unique sequence DDDDK. Recently, we have found specific activity of human enteropeptidase catalytic subunit (L-HEP) being significantly higher than that of its bovine ortholog (L-BEP). Moreover, we have discovered that L-HEP hydrolyzed several nonspecific peptidic substrates. In this work, we aimed to further characterize species-specific enteropeptidase activities and to reveal their structural basis. First, we compared hydrolysis of peptides and proteins lacking DDDDK sequence by L-HEP and L-BEP. In each case human enzyme was more efficient, with the highest hydrolysis rate observed for substrates with a large hydrophobic residue in P2-position. Computer modeling suggested enzyme exosite residues 96 (Arg in L-HEP, Lys in L-BEP) and 219 (Lys in L-HEP, Gln in L-BEP) to be responsible for these differences in enteropeptidase catalytic activity. Indeed, human-to-bovine mutations Arg96Lys, Lys219Gln shifted catalytic properties of L-HEP toward those of L-BEP. This effect was amplified in case of the double mutation Arg96Lys/Lys219Gln, but still did not cover the full difference in catalytic activities of human and bovine enzymes. To find a missing link, we studied monopeptide benzyl-arginine-ß-naphthylamide hydrolysis. L-HEP catalyzed it with an order lower K (m) than L-BEP, suggesting the monopeptide-binding S1 site input into catalytic distinction between two enteropeptidase species. Together, our findings suggest structural basis of the unique catalytic properties of human enteropeptidase and instigate further studies of its tentative physiological and pathological roles.

Enhanced soluble expression of recombinant Flavobacterium heparinum heparinase I in Escherichia coli by fusing it with various soluble partners.

Heparinase I (HepA) was originally isolated from Flavobacterium heparinum (F. heparinum) and specifically cleaves heparin/heparan sulfate in a site-dependent manner, showing great promise for producing low molecular weight heparin (LMWH). However, expressing recombinant HepA is extremely difficult in Escherichia coli because it suffers from low yields, insufficient purity and insolubility. In this paper, we systematically cloned and fused the HepA gene to the C-terminus of five soluble partners, including translation initiation factor 2 domain I (IF2), glutathione S-transferase (GST), maltose-binding protein (MBP), small ubiquitin modifying protein (SUMO) and N-utilization substance A (NusA), to screen for their abilities to improve the solubility of recombinant HepA when expressed in E. coli. A convenient two-step immobilized metal affinity chromatography (IMAC) method was utilized to purify these fused HepA hybrids. We show that, except for NusA, the fusion partners dramatically improved the soluble expression of recombinant HepA, with IF2-HepA and SUMO-HepA creating almost completely soluble HepA (98% and 94% of expressed HepA fusions are soluble, respectively), which is the highest yield rate published to the best of our knowledge. Moreover, all of the fusion proteins show comparable biological activity to their unfused counterparts and could be used directly without removing the fusion tags. Together, our results provide a viable option to produce large amounts of soluble and active recombinant HepA for manufacturing.

Crystal structure of a supercharged variant of the human enteropeptidase light chain.

The highly specific serine protease human enteropeptidase light chain cleaves the Asp4Lys recognition sequence and represents an interesting enzyme for biotechnological applications. The human enzyme shows 10 times faster kinetics compared to other animal sources but low solubility under low salt conditions, which hampers protein production and crystallization. Therefore, a supercharged variant (N6D/G21D/G22D/N142D/K210E/C112S) with increased solubility was used for crystallization. The structure (resolution, 1.9 Å) displays a typical α/ß trypsin-like serine protease-fold. The mutations introduced for protein supercharging generate larger clusters of negative potential on both sites of the active cleft but do not affect the structural integrity of the protein.