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Molecular imaging using singlechain variable fragments (scFv) of antibodies targeting cancer specific antigens have been considered a non-immunogenic approach for early diagnosis in the clinic. Usually, production of proteins is performed within Escherichia coli. Recombinant proteins are either expressed in E. coli cytoplasm as insoluble inclusion bodies, that often need cumbersome denaturation and refolding processes, or secreted toward the periplasm as soluble proteins that highly reduce the overall yield. However, production of active scFvs in their native form, without any heterologous fusion, is required for clinical applications. In this study, we expressed an anti-thymocyte differentiation antigen-scFv (Thy1-scFv) as a fusion protein with a N-terminal sequence including 3 × hexa-histidines, as purification tags, together with a Trx-tag and a S-tag for enhanced-solubility. Our strategy allowed to recover ~ 35% of Thy1-scFv in the soluble cytoplasmic fraction. An enterokinase cleavage site in between Thy1-scFv and the upstream tags was used to regenerate the protein with 97.7 ± 2.3% purity without any tags. Thy1-scFv showed functionality towards its target on flow cytometry assays. Finally, in vivo molecular imaging using Thy1-scFv conjugated to an ultrasound contrast agent (MBThy1-scFv) demonstrated signal enhancement on a transgenic pancreatic ductal adenocarcinoma (PDAC) mouse model (3.1 ± 1.2 a.u.) compared to non-targeted control (0.4 ± 0.4 a.u.) suggesting potential for PDAC early diagnosis. Overall, our strategy facilitates the expression and purification of Thy1-scFv while introducing its ability for diagnostic molecular imaging of pancreatic cancer. The presented methodology could be expanded to other important eukaryotic proteins for various applications, including but not limited to molecular imaging.

Enteropeptidase is a duodenum serine protease that triggers the activation of pancreatic enzymes by remarkably specific cleavages after lysine residues of peptidyl substrate (Asp)4-Lys. This high specific cleavage makes the enzyme a widely used biotechnological tool in laboratory researches and industrial scale. Previous studies both in small and large scales were showed low expression and miss-folding of the expressed protein. In this study, the DNA sequence encoding the light chain (catalytic subunit) of bovine enteropeptidase (EPL) was subcloned into plasmid pET-32b, downstream to the DNA encoding the fusion partner thioredoxin immediately after the EPL cleavage site. SHuffle® T7 Express was selected as an expression host due to the ability to promote proper folding and correction of the mis-oxidized bonds. Expression and purification of protein was performed, and the result of biological activity confirmed that the active EPL was obtained. Optimization of protein expression conditions was accomplished by response surface methodology for significant factors including induction temperature, duration of induction, inducer concentration and OD600 of induction. The best conditions were achieved in 1.05 mM IPTG at OD600 of 0.6 for seven h incubation at 26.5 °C, and a high level of protein expression was obtained in the optimized condition.

Bovine enterokinase light chain (EKL) is an industrially useful protease for accurate removal of affinity-purification tags from high-value biopharmaceuticals. However, recombinant expression in Escherichia coli produces insoluble inclusion bodies, requiring solubilisation, refolding, and autocatalytic activation to recover functional enzyme. Error-prone PCR and DNA shuffling of the EKL gene, T7 promoter, lac operon, ribosome binding site, and pelB leader sequence, yielded 321 unique variants after screening ~ 6500 colonies. The best variants had > 11,000-fold increased total activity in lysates, producing soluble enzyme that no longer needed refolding. Further characterisation identified the factors that improved total activity from an inactive and insoluble starting point. Stability was a major factor, whereby melting temperatures > 48.4 °C enabled good expression at 37 °C. Variants generally did not alter catalytic efficiency as measured by kcat/Km, which improved for only one variant. Codon optimisation improved the total activity in lysates produced at 37 °C. However, non-optimised codons and expression at 30 °C gave the highest activity through improved protein quality, with increased kcat and Tm values. The 321 variants were statistically analysed and mapped to protein structure. Mutations detrimental to total activity and stability clustered around the active site. By contrast, variants with increased total activity tended to combine stabilising mutations that did not disrupt the active site.

The interphotoreceptor matrix (IPM) is a specialized extracellular mesh of molecules surrounding the inner and outer segments of photoreceptor neurons. Interphotoreceptor matrix proteoglycan 1 and 2 (IMPG1 and IMPG2) are major components of the IPM. Both proteoglycans possess SEA (sperm protein, enterokinase and agrin) domains, which may support proteolysis. Interestingly, mutations in the SEA domains of IMPG1 and IMPG2 are associated with vision disease in humans. However, if SEA domains in IMPG molecules undergo proteolysis, and how this contributes to vision pathology is unknown. Therefore, we investigated SEA-mediated proteolysis of IMPG1 and IMPG2 and its significance to IPM physiology. Immunoblot analysis confirmed proteolysis of IMPG1 and IMPG2 in the retinas of wildtype mice. Point mutations mimicking human mutations in the SEA domain of IMPG1 that are associated with vision disease inhibited proteolysis. These findings demonstrate that proteolysis is part of the maturation of IMPG1 and IMPG2, in which deficits are associated with vision diseases. Further, immunohistochemical assays showed that proteolysis of IMPG2 generated two subunits, a membrane-attached peptide and an extracellular peptide. Notably, the extracellular portion of IMPG2 trafficked from the IPM around the inner segment toward the outer segment IPM by an IMPG1-dependent mechanism. This result provides the first evidence of a trafficking system that shuttles IMPG1 and IMPG2 from the inner to outer IPM in a co-dependent manner. In addition, these results suggest an interaction between IMPG1–IMPG2 and propose that mutations affecting one IMPG could affect the localization of the normal IMPG partner, contributing to the disease mechanism of vision diseases associated with defective IMPG molecules.

To discover a novel series of potent inhibitors of enteropeptidase, a membrane-bound serine protease localized to the duodenal brush border, 4-guanidinobenzoate derivatives were evaluated with minimal systemic exposure. The

BackgroundInhibiting enteropeptidase, a gut serine protease regulating protein digestion, suppresses food intake and ameliorates obesity and diabetes in mice. However, the effects of enteropeptidase inhibition on kidney parameters are largely unknown. Here, we evaluated the chronic effects of an enteropeptidase inhibitor, SCO-792, on kidney function, albuminuria and kidney pathology in spontaneously hypercholesterolaemic (SHC) rats, a rat chronic kidney disease (CKD) model.MethodsSCO-792, an orally available enteropeptidase inhibitor, was administered [0.03% and 0.06% (w/w) in the diet] to 20-week-old SHC rats showing albuminuria and progressive decline in glomerular filtration rate (GFR) for five weeks. The effects of SCO-792 and the contribution of amino acids to these effects were evaluated.ResultsSCO-792 increased the faecal protein content, indicating that SCO-792 inhibited enteropeptidase in SHC rats. Chronic treatment with SCO-792 prevented GFR decline and suppressed albuminuria. Moreover, SCO-792 improved glomerulosclerosis and kidney fibrosis. Pair feeding with SCO-792 (0.06%) was less effective in preventing GFR decline, albuminuria and renal histological damage than SCO-792 treatment, indicating the enteropeptidase-inhibition-dependent therapeutic effects of SCO-792. SCO-792 did not affect the renal plasma flow, suggesting that its effect on GFR was mediated by an improvement in filtration fraction. Moreover, SCO-792 increased hydrogen sulphide production capacity, which has a role in tissue protection. Finally, methionine and cysteine supplementation to the diet abrogated SCO-792-induced therapeutic effects on albuminuria.ConclusionsSCO-792-mediated inhibition of enteropeptidase potently prevented GFR decline, albuminuria and kidney fibrosis; hence, it may have therapeutic potential against CKD.

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 (kinact/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 A, 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.

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-ASP

Aims Enteropeptidase is a serine protease localized on the duodenal brush border that catalyzes the conversion of inactive trypsinogen into active trypsin, thereby regulating protein breakdown in the gut. We evaluated the effects of SCO‐792, a novel enteropeptidase inhibitor, in mice. Materials and methods In vivo inhibition of enteropeptidase was evaluated via an oral protein challenge. Pharmacological effects were evaluated in normal mice, in diet‐induced obese (DIO) mice and in obese and diabetic ob/ob mice. Results A single oral administration of SCO‐792 inhibited plasma branched‐chain amino acids (BCAAs) in an oral protein challenge test in mice, indicating in vivo inhibition of enteropeptidase. Repeated treatment with SCO‐792 induced reduction in food intake and decrease in body weight in DIO and ob/ob mice. Plasma FGF21 levels were increased in SCO‐792‐treated DIO mice, an observation that was probably independent of reduction in food intake. Hyperglycaemia was markedly improved in SCO‐792‐treated ob/ob mice. A hyperinsulinaemic‐euglycaemic clamp study revealed improved muscle insulin sensitivity in SCO‐792‐treated ob/ob mice. SCO‐792 also improved plasma and liver lipid profiles and decreased plasma alanine transaminase, suggesting a potential treatment for liver diseases. Dietary supplementation with essential amino acids attenuated the effect of SCO‐792 on reduction in food intake and decrease in body weight in normal mice, suggesting a pivotal role for enteropeptidase in these biological phenomena. Conclusions SCO‐792 inhibited enteropeptidase in vivo, reduced food intake, decreased body weight, increased insulin sensitivity, improved glucose and lipid control, and ameliorated liver parameters in mouse models with obesity and/or diabetes. SCO‐792 may exhibit similar effects in patients.

A novel series of guanidinebenzoate enteropeptidase and trypsin dual inhibitors has been discovered and SAR studies were conducted. Optimization was focused on improving properties for gut restriction, including increased aqueous solubility, lower cellular permeability, and reduced oral bioavailability. Lead compounds were identified with efficacy in a mouse fecal protein excretion study.

Aim To examine the effects of an enteropeptidase inhibitor, SCO‐792, on kidney function in rats. Materials and Methods The pharmacological effects of SCO‐792 were evaluated in Wistar fatty (WF) rats, a rat model of diabetic kidney disease (DKD). Results Oral administration of SCO‐792 increased faecal protein content and improved glycaemic control in WF rats. SCO‐792 elicited a rapid decrease in urine albumin‐to‐creatinine ratio (UACR). SCO‐792 also normalized glomerular hyperfiltration and decreased fibrosis, inflammation and tubular injury markers in the kidneys. However, pioglitazone‐induced glycaemic improvement had no effect on kidney variables. Dietary supplementation of amino acids (AAs), which bypass the action of enteropeptidase inhibition, mitigated the effect of SCO‐792 on UACR reduction, suggesting a pivotal role for enteropeptidase. Furthermore, autophagy activity in the glomerulus, which is impaired in DKD, was elevated in SCO‐792‐treated rats. Finally, a therapeutically additive effect on UACR reduction was observed with a combination of SCO‐792 with irbesartan, an angiotensin II receptor blocker. Conclusions This study is the first to demonstrate that enteropeptidase inhibition is effective in improving disease conditions in DKD. SCO‐792‐induced therapeutic efficacy is likely to be independent of glycaemic control and mediated by the regulation of AAs and autophagy. Taken together with a combination effect of irbesartan, SCO‐792 may be a novel therapeutic option for patients with DKD.

We propose a yeast display-based system for screening of proteolytic enzyme libraries that utilizes substrate protein adsorbed on the yeast cell surface and containing a desired cleavage sequence. Specific cleavage of the substrate protein releases its biotin-binding center. The cells carrying the target proteinase can be selected by cytofluorometry due to interaction with biotinylated fluorescent protein. Using human enterokinase light chain as the model proteinase we showed that the proposed screening system highly effectively selects the proteolytic enzymes with preset specificity.

In Japan, there are two species of scorpions, Madara scorpion (Isometrus maculatus) and Yaeyama scorpion (Liocheles australasiae), and both of them are living in Yaeyama island. It has been shown that Liocheles australasiae has venom including β-toxin acting on K+-channels (β-KTx) (Juichi et al., 2018) [1]. Interestingly, LaIT2, one of the toxins found in the venom of Liocheles australasiae, displays the virulence for insects but almost not for mammals. Until now, molecular mechanism of the functional specificity of LaIT2 is unknown. To clear this issue, we tried to establish the overexpression system of LaIT2 in Rosetta-gami B (DE3) pLysS, which have trxB/gor mutations to induce the disulfide bond formation. In this study, we have succeeded to overexpress the recombinant LaIT2 (rLaIT2) as a thioredoxin (Trx)-tagged protein, and established the purification protocol with Ni2+-NTA column chromatography, enterokinase digestion, and HPLC. We succeeded to obtain approximately 0.5 mg of rLaIT2 from the E. coli cells cultured in 1 L of M9 culture medium. Intramolecular disulfide bonding pattern of rLaIT2 was identified by endopeptidase fragmentation and mass spectrometry. rLaIT2 showed insecticidal activity and antimicrobial activity, and these are almost identical to those of natural LaIT2. 1H–15N HSQC spectrum of 15N-labeled rLaIT2 indicated that the rLaIT2 has a stable conformation.

Human acidic fibroblast growth factor (haFGF) is a multifunctional protein involved in regulating a wide range of cellular processes. As a potent therapeutic agent, it is highly desirable to produce recombinant haFGF (r-haFGF) at low cost. However, the complex structure and formation of aggregation confines its high-level soluble expression and functional form. Herein, to produce r-haFGF efficiently in E. coli, we devised a novel soluble expression and cost-effective purification approach based on fusion with Scl2-M (a novel modified collagen-like protein) for the first time. By using this strategy, more than 95% of the Scl2-M-haFGF fusion protein was highly expressed in soluble form and the expression level of targeted fusion protein in shake flasks and 5-L fermenter was 0.42 g/L and 2.28 g/L, respectively. Subsequently, the recombinant Scl2-M-haFGF was readily purified through a facile process of acid precipitation and subjected to enterokinase (EK) cleavage. After Scl2-M cleavage, tag-free r-haFGF was further purified using ion-exchange chromatography. The recovery rate of the whole purification process attained 34.2%. Furthermore, the resulting high-purity (96.0%) r-haFGF was prepared by freeze-drying as a final product, and its bioactivity was confirmed to potentiate the proliferation of L929 and BALB-3T3 fibroblasts. Overall, our developed method has the potential for the massive production of the r-haFGF in the future.

Enteropeptidase, localized into the duodenum brush border, is a key enzyme catalyzing the conversion of pancreatic trypsinogen proenzyme to active trypsin, thereby regulating protein digestion and energy homeostasis. We report the discovery and pharmacological profiles of SCO‐792, a novel inhibitor of enteropeptidase. A screen employing fluorescence resonance energy transfer was performed to identify enteropeptidase inhibitors. Inhibitory profiles were determined by in vitro assays. To evaluate the in vivo inhibitory effect on protein digestion, an oral protein challenge test was performed in rats. Our screen identified a series of enteropeptidase inhibitors, and compound optimization resulted in identification of SCO‐792, which inhibited enteropeptidase activity in vitro, with IC 50 values of 4.6 and 5.4 nmol/L in rats and humans, respectively. In vitro inhibition of enteropeptidase by SCO‐792 was potentiated by increased incubation time, and the calculated K inact/KI was 82 000/mol/L s. An in vitro dissociation assay showed that SCO‐792 had a dissociation half‐life of almost 14 hour, with a calculated k off rate of 0.047/hour, which suggested that SCO‐792 is a reversible enteropeptidase inhibitor. In normal rats, a ≤4 hour prior oral dose of SCO‐792 effectively inhibited plasma elevation of branched‐chain amino acids in an oral protein challenge test, which indicated that SCO‐792 effectively inhibited protein digestion in vivo. In conclusion, our new screen system identified SCO‐792 as a potent and reversible inhibitor against enteropeptidase. SCO‐792 slowly dissociated from enteropeptidase in vitro and inhibited protein digestion in vivo. Further study using SCO‐792 could reveal the effects of inhibiting enteropeptidase on biological actions.

Background Studying proteins and enzymes involved in important biological processes in the Aedes aegypti mosquito is limited by the quantity that can be directly isolated from the mosquito. Adding to this difficulty, digestive enzymes (midgut proteases) involved in metabolizing blood meal proteins require a more oxidizing environment to allow proper folding of disulfide bonds. Therefore, recombinant techniques to express foreign proteins in Escherichia coli prove to be effective in producing milligram quantities of the expressed product. However, with the most commonly used strains having a reducing cytoplasm, soluble expression of recombinant proteases is hampered. Fortunately, new E. coli strains with a more oxidizing cytoplasm are now available to ensure proper folding of disulfide bonds. Results Utilizing an E. coli strain with a more oxidizing cytoplasm (SHuffle® T7, New England Biolabs) and changes in bacterial growth temperature has resulted in the soluble expression of the four most abundantly expressed Ae. aegypti midgut proteases (AaET, AaSPVI, AaSPVII, and AaLT). A previous attempt of solubly expressing the full-length zymogen forms of these proteases with the leader (signal) sequence and a modified pseudo propeptide with a heterologous enterokinase cleavage site led to insoluble recombinant protein expression. In combination with the more oxidizing cytoplasm, and changes in growth temperature, helped improve the solubility of the zymogen (no leader) native propeptide proteases in E. coli. Furthermore, the approach led to autocatalytic activation of the proteases during bacterial expression and observable BApNA activity. Different time-points after bacterial growth induction were tested to determine the time at which the inactive (zymogen) species is observed to transition to the active form. This helped with the purification and isolation of only the inactive zymogen forms using Nickel affinity. Conclusions The difficulty in solubly expressing recombinant proteases in E. coli is caused by the native reducing cytoplasm. However, with bacterial strains with a more oxidizing cytoplasm, recombinant soluble expression can be achieved, but only in concert with changes in bacterial growth temperature. The method described herein should provide a facile starting point to recombinantly expressing Ae. aegypti mosquito proteases or proteins dependent on disulfide bonds utilizing E. coli as a host. Electronic supplementary material The online version of this article (10.1186/s12858-018-0101-0) contains supplementary material, which is available to authorized users.

Enteropeptidase is a transmembrane serine protease localized in the lumen of the duodenum that acts as a key enzyme for protein digestion. SCO-792 is an orally available enteropeptidase inhibitor that has been reported to have therapeutic effects on obesity and diabetes in mice. However, the mechanism underlying the therapeutic effect of SCO-792 has not yet been fully elucidated. In this study, we evaluated the role of gut microbiota on SCO-792-induced body weight (BW) reduction in high-fat diet-induced obese (DIO) mice. Chronic administration of SCO-792 substantially decreased BW and food intake in DIO mice. While the pair-fed study uncovered food intake-independent mechanisms of BW reduction by SCO-792. Interestingly, antibiotics-induced microbiota elimination in the gut canceled SCO-792-induced BW reduction by nearly half without affecting the anorectic effect, indicating the involvement of gut microbiota in the anti-obesity mechanism that is independent of food intake reduction. Microbiome analysis revealed that SCO-792 altered the gut microbiota composition in DIO mice. Notably, it was found that the abundance of Firmicutes decreased while that of Verrucomicrobia increased at the phylum level. Increased abundance of Akkermansia muciniphila, a bacterium known to be useful for host metabolism, was observed in SCO-792-treated mice. Fecal metabolome analysis revealed increased amino acid levels, indicating gut enteropeptidase inhibition. In addition, SCO-792 was found to increase the level of short-chain fatty acids, including propionate, and bile acids in the feces, which all help maintain gut health and improve metabolism. Furthermore, it was found that SCO-792 induced the elevation of colonic immunoglobulin A (IgA) concentration, which may maintain the microbiota condition, in DIO mice. In conclusion, this study demonstrates the contribution of microbiota to SCO-792-induced BW reduction. Enteropeptidase-mediated regulation of microbiota, enterobacterial metabolites, and IgA in the gut may coordinately drive the therapeutic effects of SCO-792 in obesity.

Salmonids have four subtypes of insulin-like growth factor binding protein (IGFBP)-1, termed -1a1, -1a2, -1b1 and 1b2, owing to teleost- and a lineage-specific whole-genome duplications. We have previously produced recombinant proteins of masu salmon IGFBP-1a1 and -1b2 and conducted functional analysis. To further characterize salmonid-specific IGFBP-1s, we cloned cDNAs encoding mature proteins of IGFBP-1a2 and -1b1 from the liver of masu salmon (Oncorhynchus masou). IGFBP-1a2 and -1b1 shared a 56% amino acid sequence homology whereas their homologies with their counterparts (i.e. -1a1 and -1b2) were 77% and 82%, respectively. We next expressed recombinant masu salmon (rs) IGFBP-1a2 and -1b1 with fusion partners thioredoxin (Trx) and a His-tag using the pET-32a(+) vector system in Escherichia coli. Trx.His.rsIGFBP-1s were detected in the insoluble faction, solubilized in a buffer containing urea, and isolated by Ni-affinity chromatography. They were refolded by dialysis and cleaved from the fusion partners by enterokinase. rsIGFBP-1a2 and -1b1 were purified by reversed-phase high performance liquid chromatography. Purified rsIGFBP-1a2 and -1b1 had the ability to bind digoxigenin-labeled human IGF-I on ligand blotting. We then examined the effects of rsIGFBP-1a1, -1a2, -1b1 and -1b2 in combination with human IGF-I on growth hormone (GH) release from cultured pituitary cells of masu salmon. IGF-I alone reduced GH release while the addition of rsIGFBP-1a1, -1b1 or -1b2, but not rsIGFBP-1a2, diminished the suppressive effect of IGF-I. Addition of rsIGFBP-1s without IGF-I had no effect on GH release. These results show that rsIGFBP-1b1, along with rsIGFBP-1a1 and -1b2, inhibits IGF-I action on the pituitary in masu salmon. The lack of the effect by rsIGFBP-1a2 suggests that salmon IGFBP-1 subtypes underwent subfunction partitioning and have different degrees of IGF-inhibitory action.

The domestic ferret (Mustela putorius furo) recently emerged as a novel model for human pancreatic diseases. To investigate whether the ferret would be appropriate to study hereditary pancreatitis associated with increased trypsinogen autoactivation, we purified and cloned the trypsinogen isoforms from the ferret pancreas and studied their functional properties. We found two highly expressed isoforms, anionic and cationic trypsinogen. When compared to human cationic trypsinogen (PRSS1), ferret anionic trypsinogen autoactivated only in the presence of high calcium concentrations but not in millimolar calcium, which prevails in the secretory pathway. Ferret cationic trypsinogen was completely defective in autoactivation under all conditions tested. However, both isoforms were readily activated by enteropeptidase and cathepsin B. We conclude that ferret trypsinogens do not autoactivate as their human paralogs and cannot be used to model the effects of trypsinogen mutations associated with human hereditary pancreatitis. Intra-pancreatic trypsinogen activation by cathepsin B can occur in ferrets, which might trigger pancreatitis even in the absence of trypsinogen autoactivation.

Trypsin-like serine proteases are essential in physiological processes. Studies have shown that N-glycans are important for serine protease expression and secretion, but the underlying mechanisms are poorly understood. Here, we report a common mechanism of N-glycosylation in the protease domains of corin, enteropeptidase and prothrombin in calnexin-mediated glycoprotein folding and extracellular expression. This mechanism, which is independent of calreticulin and operates in a domain-autonomous manner, involves two steps: direct calnexin binding to target proteins and subsequent calnexin binding to monoglucosylated N-glycans. Elimination of N-glycosylation sites in the protease domains of corin, enteropeptidase and prothrombin inhibits corin and enteropeptidase cell surface expression and prothrombin secretion in transfected HEK293 cells. Similarly, knocking down calnexin expression in cultured cardiomyocytes and hepatocytes reduced corin cell surface expression and prothrombin secretion, respectively. Our results suggest that this may be a general mechanism in the trypsin-like serine proteases with N-glycosylation sites in their protease domains.

Enterokinase is an ideal tool protease for cleaving fusion proteins in genetic engineering. The bovine enterokinase light chain (bEK

Lunasin is a novel promising health-beneficial peptide derived from soybean. However, the application of lunasin is limited by its high cost. In this study, we developed a successful protocol for expression of a dimer formation protein containing 4 tandem repeated lunasin analogs (lunasin-4) in Pichia pastoris. The expression level at the optimal condition (initial pH 7.0, 1.0% final methanol concentration and induction for 72 h at 26 °C) was 0.24 mg/mL cell-free broth. Lunasin analog, obtained from purified lunasin-4 protein through enterokinase digestion and ultrafiltration, significantly decreased (p 0.05) the release of nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages in a dose-dependent manner. In addition, intracellular signaling array analysis demonstrated down-regulated levels of phosphorylated Akt, mechanistic target of rapamycin (mTOR) and p70 s6 kinase (p70s6k) and an up-regulated level of glycogen synthase kinase-3β (GSK-3β) after lunasin analog treatment. These results suggest that lunasin analog exerted anti-inflammatory activities in LPS-stimulated RAW264.7 cells partly via inhibiting the activation of Akt/mTOR/p70s6k signaling pathway. In conclusion, this study provides a potential strategy for recombinant production of bioactive lunasin in industry.

Previously, we reported that the medaka testis abundantly expresses the mRNA for trypsinogen, which is a well-known pancreatic proenzyme that is secreted into and activated in the intestine. Currently, we report our characterization of the medaka trypsin using a recombinant enzyme and show that this protein is a serine protease that shares properties with trypsins from other species. Two polypeptides (28- and 26-kDa) were detected in the testis extracts by Western blot analysis using antibodies that are specific for medaka trypsinogen. The 28-kDa polypeptide was shown to be trypsinogen (inactive precursor), and the 26-kDa polypeptide was shown to be trypsin (active protease). We did not detect enteropeptidase, which is the specific activator of trypsinogen, in the testis extract. Immunohistochemical analyses using the same trypsinogen-specific antibody produced a strong signal in the spermatogonia and spermatozoa of the mature medaka testis. Substantial staining was found with spermatocytes, whereas extremely weak signals were observed with spermatids. In vitro incubation of testis fragments with the trypsinogen antibody strongly inhibited the release of sperm from the testis into the medium. Trypsin activity was detected in sperm extracts using gelatin zymographic analysis. Immunocytochemistry showed that trypsinogen and trypsin were localized to the cell membranes surrounding the sperm head. Collectively, these results suggest that trypsin plays an important role in the testis function of the medaka.

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 β-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.

Enteropeptidase (EC 3.4.21.9) is the glycoprotein enzyme in the small intestine that triggers the activation of the zymogens in pancreatic juice by converting trypsinogen into trypsin. Because of its physiological significance, there have been many studies on the expression, purification, and characterization of enteropeptidase from different species. The baculovirus expression system has been commonly used in research communities and scientific industries for the production of high levels of recombinant proteins, which require posttranslational modifications for functional activity. In the present study, we isolated bovine enteropeptidase catalytic subunit gene from Bos taurus indicus (GenBank accession no. KC756844), and cloned it in pFast Bac HT "A" baculovirus expression donor vector, under the polyhedrin promoter. Recombinant bovine enteropeptidase was expressed in SF-9 insect cells with high expression levels. Recombinant enteropeptidase was purified using Ni-NTA affinity chromatography. A 6-mg quantity of pure active protein was obtained from 100 mL culture using this approach. Its activity and kinetic parameters were determined by cleavage of its fluorogenic substrate Gly-(Asp) 4-Lys-β-naphthylamide. The recombinant bovine enteropeptidase showed a K m value of 0.75 ± 0.02 mM with K cat 25 ± 1 s.

Dear Editor, Enteropeptidase (enterokinase, EC 3.4.21.9) is a serine protease, which shows a specific cleavage of its substrates at the C-terminal of the recognition site (Asp)4Lys (Zheng et al., 2009). Because of the unique specificity, enteropeptidase could be used as a tool for the production of recombinant fusion proteins. Especially the recombinant enteropeptidase light chain (EPL), which contains a catalytic domain, is of large interest to be applied in biopharmaceutical industry (Lu et al., 1997). Enteropeptidase has been cloned from several sources, including bovine (Kitamoto et al., 1994), porcine (Matsushima et al., 1994), humans (Kitamoto et al., 1995), mouse (Yuan et al., 1998), and rat (Yahagi et al., 1996). For the high availability, the recombinant bovine enteropeptidase light chain (BEPL) is now most commonly used. However, it is known that BEPL does not exhibit high stringency in its specificity for the canonical target sequence D4K (Liew et al., 2007). For instance, Shahravan et al. showed that BEPL also cleaved at unexpected DR and SR sites in AhR6-C/EBP protein (Shahravan et al., 2008). Recently, the enteropeptidase from medaka had been reported to be a more effective tool, because of its much stricter specific for the D4K sequence compared to its mammalian counterparts (Ogiwara and Takahashi, 2007). Therefore, we sought to solve the crystal structure of the light chain of medaka enteropeptidase (MEPL), and gain insights into the determinants for its stricter specificity. MEPL shares high sequence similarity with other EPL classes, which have been structurally identified in previous reports (Fig. S1). Therefore, the crystal structure of MEPL was determined by molecular replacement using the bovine enteropeptidase light chain (PDB entry 1EKB, 53.7% amino acid identity) as the search model and refined to 2.0 A resolution. The structure of MEPL displays a typical α/β trypsin—like serine protease fold (Fig. 1A). It consists of two six-stranded β-barrels (β1–β6 and β7–β12), either of which makes up about one half of the entire molecule. Both β-barrels are arranged in a Greek-key-pattern containing α-helices at the middle of each barrel with a third α-helix located at the C-terminus. The surface potentials of MEPL reveal an equal distribution of charged amino acids on protein surface although the region near the active center has a predominantly negative potential (Fig. 1B). Figure 1 Structure of MEPL. (A) Overall structure of MEPL. The structure of MEPL is shown as ribbon diagrams in two orientations: Front view, looking into the catalytic center; side view, besides the catalytic center. The MEPL molecules are colored as blue-white ... Superimposing of MEPL with the bovine enteropeptidase light chain resulted in an rmsd of 0.79 A for the Ca coordinates (Fig. 1C). The differences are mainly located in the loop regions, such as the so-called ‘131-loop’ that connects strand β7 and β8. MEPL also contains, relative to bovine enzyme, an additional small 310-helix between the strand β4 and β5. Furthermore, the catalytic triad of the unliganded medaka enteropeptidase superimposes well with the bovine enzyme in complex with a trypsinogen-activation peptide analogue (Lu et al., 1999). However, in secondary structure, the active centers do not show any obvious differences between the two enzymes, which intimates that the stricter specificity of MEPL may depend on the distinct amino acid sequence and unique intramolecular interactions. The comparison of the catalytic centers between MEPL, BEPL and HEPL (human enteropeptidase light chain, PDB entry 4DGJ, amino acid identity 55%), provide an understanding of the mechanism of substrate specificity functioned by these different enzymes (Fig. 2A and ​and22B). Figure 2 Comparison of the catalytic center in MEPL, BEPL and HEPL, and specific assay of MEPL variants. (A and B) View of detailed two regions considered as playing crucial role in the specificity mechanism. Key residues are shown as colored sticks (blue-white ... In the structure of BEPL with a trypsinogen-activation peptide (VD4K), the side chain of Lys-P1 inserts deeply into a specific pocket, at the bottom of which Asp181 neutralizes the terminal amino group (amino acid residues of peptidyl substrates customarily are numbered P1, P2, P3, etc. (Schechter and Berger, 1967)). The structure of this cleft determinates that the P1 site of substrates could only be lysine or arginine (Lu et al., 1999). This specific pocket consists of three parts: the strand β11, the so called “174-loop” that connects strand β9 and β10, and the “208-loop” that connects strand β11 and β12. The main differences locate at the N-terminus of “208-loop”, while the sequences of strand β11 and “174-loop” are highly conservative. Compared with BEPL and HEPL, MEPL shows much less activity for the peptide substrates with arginine at P1 site, which indicates its restriction for the entrance of arginine residues. And according to the structural details, we supposed that the variations of MEPLH24, MEPLE136, MEPLV209, MEPLG210 and MEPLR213 may play curial role in the tension changes of the loop chains around the pocket of catalytic center. In MEPL structure, MEPLE136 makes three hydrogen bonds with MEPLR213, while there are no similar interactions in BEPL because it is BEPLL213, BEPLY136 in these positions as well as the same situation for the HEPL structure (Fig. 2A). Although the interactions are a little far distance from the catalytic center (8 A), these hydrogen bonds may help anchoring the “208-loop”, restricting the electron density size of substrate. In addition, MEPLV209 and MEPLG210 with short side chains are covered and fixed by the side chains of four residues (MEPLE136, MEPLE163, MEPLR213 and MEPLR216), reducing the mobility of “208-loop” and strand β11 in MEPL. However, the amino acid residues at the same positions in BEPL and HEPL are exposed on the surface of the proteins, and therefore possess higher flexibility (Figs. 2A and S2). Moreover, we observed that the imidazole group of MEPLH24 could form a hydrogen bond with the main chain oxygen atom of MEPLG185, which could help improving the structural rigidity of “174-loop” (Fig. 2B). Nevertheless, there are no such interactions in BEPL and HEPL because it is BEPLQ24 and HEPLL24 at this position, respectively. Over all, the unique residue interactions in MEPL may reduce the flexibility of the specific pocket, restricting the entrance of arginine residue which has larger side chain. In order to study the impacts of these residues on MEKL-substrate interaction, 3 residues in MEPL were replaced by those in BEPL respectively (H24Q, E136Y and R213L), and a combined mutant E136Y/R213L was built. Then, the specific activities of the four variants were assayed by using the specific substrate GD4K-βNA and the non-specific substrates Boc-E(OBzl)-AR-MCA and Z-FR-MCA. The results showed that three mutants (H24Q, R213L and E136Y/R213L) exhibited significantly increased activity for the MCA-containing substrates, while retaining GD4K-βNA hydrolyzing activity (Fig. 2C and ​and2D).2D). This result indicated the importance of the two interactions (H24-G185 and E136-R213) for the specificity of enteropeptidase. Nevertheless, these mutations did not raise the activities of MEPL on unspecific substrates to the level of BEPL, which meant there should be other facts affecting the substrate-selectivity of MEPL. The low enzyme activity of mutant E136Y for both GD4K-βNA and the MCA-containing substrates meant that the amino acids in position 136 might affect the activity of enteropeptidase. And it was also been provided by the kinetic studies (Table S3). Taken together, our data on cleavage of peptide substrates, faithfully supported by crystal structures, present an extraordinary example of fine adjustment of enzyme mechanism. Of note, this finding can be directly applied in the reengineering of other enteropeptidases, for instance BEPL and HEPL, to improve the specificity.

Human growth hormone (hGH) is the major and important hormone component of human being. At present, hGH for clinical uses is mostly produced in Escherichia coli, which requires costly denaturation and refolding to recover functionality. To obtain long-term bioactive hormone, we used hGH as a foreign gene and constructed a recombinant plasmid pJS700-hGH which carries a recombinant gene cotC-hgh with an enterokinase site under the control of cotC promoter. Plasmid pJS700-hGH was transformed into Bacillus subtilis by double crossover and an amylase-inactivated mutant was produced. After spore formation, Western blot and fluorescence immunoassay were used to monitor hGH surface expression on spores. Oral administration to silkworm with spores displaying hGH further showed that the recombinant spores may have potential ability to be digested and absorbed into the silkworm's hemolymph due to both the resistant characters of spores and the addition of enterokinase site.

Highlights • Histidine-tagged bovine enterokinase was refolded from bacterial inclusion bodies. • Refolding yields satisfy high demands of protein crystallography projects. • Enterokinase specifically cleaved artificial propeptides from target proteins., 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.

A duodenase, a protease structurally related to human cathepsin G, was found earlier in bovine duodenal mucosa. It was demonstrated that under the influence of duodenase an enteropeptidase zymogen is activated in vitro showing the possible participation of duodenase in the cascade of activation of digestive enzymes. To identify a duodenase functional analog in human duodenum, an immunofluorescence study of duodenal mucosa was conducted by confocal microscopy using antibodies to human cathepsin G and to bovine duodenase. The previously unknown place of synthesis and secretion of cathepsin G - Paneth cells located at the bottom of Lieberkuhn crypts - was revealed. Binding of cathepsin G-specific antibodies in a rough endoplasmic reticulum zone and in the cryptal duct was observed. Duodenase-specific immunofluorescence but not that of cathepsin G was found in the epitheliocytes and secretory ducts of Brunner's glands, which are characteristic sites of duodenase biosynthesis in cattle. Binding of CD14-specific antibodies in the Brunner's glands, where the antibodies co-localized with the antibodies to duodenase, was also demonstrated. These data indicate the presence of a protein immunologically similar to duodenase in the human duodenal mucosa. Our study demonstrated the absence of its co-localization with cathepsin G in Brunner's glands.

Encapsulation of active proteins in the hydrophilic core of vesicular liposomes is important for developing a therapeutic protein carrier system. The efficiency of liposomal encapsulation of proteins is generally low. A better understanding of the fundamental mechanisms of encapsulation is needed to increase this efficiency. In this study we investigated the effects of operating parameters such as phospholipid concentration, buffer pH and ionic strength, protein size and surface charge, and liposome size on the enzyme encapsulation yield. Four model enzymes of different molecular weights and isoelectric points (trypsin, horseradish peroxidase, enterokinase and hyaluronidase) were encapsulated into three different sized liposomes (125 nm, 194 nm, and 314 nm in mean diameter). Relatively inert and neutral DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) was used as the main phospholipid in the liposomes. Size exclusion chromatography was used to separate the enzyme-encapsulated liposomes from the free enzyme, and the encapsulation yield was determined from the peak area. The encapsulation yield was generally low ranging from ca. 5% to 20%, and did not depend much on the molecular weight of the enzyme encapsulated. Larger liposomes had higher encapsulation yields. The electrostatic interaction between the phospholipid and enzyme was the most significant parameter in determining the encapsulation yield. Thus adjusting buffer pH and ionic strength and adding charged phospholipids to the liposome preparation to impart electric charge to the lipid bilayer could significantly improve the yield. This approach can be used to optimize the liposomal encapsulation of clinically significant proteins.

Bioluminescence resonance energy transfer (BRET) has gained favors in recent years as a detection technology for protease activity due to its extreme reliability, high sensitivity and low intrinsic backgrounds. Because of the sensitivity of the donors, substrates and the acceptors, it is expected that BRET systems are sensitive to buffer environments. However, no systematic study has been reported on how buffer components would affect the BRET ratio, and thus affect the determination of protease activity based on BRET. We present here that several environmental factors, including buffer agents, pH and divalent metal ions, influenced BRET ratio significantly, when humanized Gaussia luciferase (hGluc) was utilized as the donor and enhanced yellow fluorescence protein (EYFP) as the acceptor. Based on these findings, an enhancing solution was optimized to improve the performance of the BRET sensor for analysis of enterokinase activity in vitro, resulting in 10-fold and 7-fold improvement of the sensitivity and the detection limit, respectively. We anticipate the system will be applicable for improving performance of other in vitro BRET protease sensors, especially when the optimal conditions for protease activity would severely affect the BRET signal.

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-β-naphthylamide hydrolysis. L-HEP catalyzed it with an order lower Km 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.

Although they are often exploited to facilitate the expression and purification of recombinant proteins, every affinity tag, whether large or small, has the potential to interfere with the structure and function of its fusion partner. For this reason, reliable methods for removing affinity tags are needed. Only enzymes have the requisite specificity to be generally useful reagents for this purpose. In this review, the advantages and disadvantages of some commonly used endo- and exoproteases are discussed in light of the latest information.

Enteropeptidase is a serine protease secreted by the pancreas and converts inactive trypsinogen to active trypsin. Enteropeptidase cleaves the C-terminal end of the substrate recognition sequence Asp-Asp-Asp-Asp-Lys (D(4)K). The assay for enteropeptidase has utilized GD(4)K-conjugated 2-naphthylamine (GD(4)K-NA) as a fluorogenic probe over the last 30 years. However, no other D(4)K-conjugated fluorogenic substrates of enteropeptidase have been reported. Furthermore, naphthalene is known as carcinogenic to humans. In this study, we used shift in the emission spectrum of GD(4)K-conjugated 7-amino-4-methylcoumarin (GD(4)K-AMC) as a fluorogenic method to measure enteropeptidase activity. The kinetic analysis revealed that enteropeptidase has a K(M) of 0.025 mM and a k(cat) of 65 sec(-1) for GD(4)K-AMC, whereas it has a K(M) of 0.5 to 0.6 mM and a k(cat) of 25 sec(-1) for GD(4)K-NA. The optimum pH of GD(4)K-AMC hydrolysis was pH 8.0. Our data indicate that GD(4)K-AMC is more suitable as a substrate for enteropeptidase than GD(4)K-NA.

Ribonuclease inhibitor (RI) is a 50-kDa cytosolic scavenger of pancreatic-type ribonucleases which inhibits ribonucleolytic activity. Expression of recombinant RI is extremely difficult to reach high levels in soluble form in the cytoplasm of Escherichia coli. Here, we utilized five N-terminal fusion partners to improve the soluble expression of RI. Among these five fusion partners which have been screened, maltose-binding protein (MBP), N-utilization substance A (NusA) and translation initiation factor 2 domain I (IF2) have greatly improved the soluble expression level of recombinant murine RI under the drive of T7 promoter, while glutathione S-transferase (GST) and small ubiquitin modifying protein (SUMO) were much less efficient. All these RI-fusion proteins remained to be highly active in inhibiting RNase A activity. Furthermore, all fusion tags can be efficiently removed by enterokinase digestion to generate native RI which results the highest yield to date (>30mg of native RI per liter culture). And a convenient two-step immobilized metal affinity chromatography (IMAC) method has been implemented in our study, comparing with the traditional RNase A affinity chromatography method.

Enterokinase light chain (EKL) is a serine protease that recognizes Asp-Asp-Asp-Asp-Lys (D(4)K) sequence and cleaves the C-terminal peptide bond of the lysine residue. The utility of EKL as a site-specific cleavage enzyme is hampered by sporadic cleavage at other sites than the canonical D(4)K recognition sequence. In order to produce more site-specific EKL, we have generated several EKL mutants in E. coli with substitutions at Tyr174 and Lys99 using PDI (protein disulfide isomerase) fusion system. Substitution of Tyr174 by basic residues confers higher specificity on EKL. The production of EKL with higher specificity could widen the utility of EKL as a site-specific cleavage enzyme to produce various recombinant proteins with therapeutic or industrial values.

In this study, human globular adiponectin-glucagon-like peptide-1 analog (gAd-GLP-1-A) fu- sion protein was expressed and its glucose-lowering effect was measured in vivo. We con- structed a prokaryotic expression vector PET28a-gAd-GLP-1-A and transformed the vector into Escherichia coli BL21 (DE3). A recombinant fusion protein of about 25KD was expressed from BL21 (DE3) cells after isopropylthio--D-galactoside induction. This protein was N-terminal His-tagged gAd-GLP-1-A fusion protein. Most of the protein was expressed in inclusion body. The fusion protein in inclusion body was purified by using High-Affinity Nickel Iminodiacetic Acid Resin and refolded in urea gradient refolding buffer. The refolded protein was incubated with enterokinase to remove the N-terminal His-tag. The fusion protein without His-tag is gAd-GLP-1-A fusion protein, which exhibited significant glu- cose-lowering effect in diabetic mice.

The extracellular loop 2 of a tetraspanin from Schistosoma japonicum (Sj-TSP-2) is homologous to Schistosoma mansoni TSP-2. In our initial study, Sj-TSP-2 is an identical antigen against schistosomiasis caused by S. japonicum. Through the pET32 vector system and nickel (Ni)-absorbed chelating Sepharose, Sj-TSP-2 was expressed and purified as a soluble fusion constructed with an N-terminal thioredoxin-His(6)-EK protease site tag (Trx-TSP-2). In phosphate buffer (PB) with a low concentration of imidazole, the Trx-TSP-2 fusion protein was efficiently cleaved by enterokinase (EK). Sj-TSP-2 was isolated and enriched using cobalt (Co)-absorbed chelating Sepharose and HiTrap SP column. Character of the protein was analyzed via animal experiments and then clinical trials. The purification approach yielded pure Sj-TSP-2, which will provide feasible advices for discovering vaccines against schistosomiasis.

Matriptase is a transmembrane serine protease that is strongly expressed in epithelial cells. The single-chain zymogen of matriptase is considered to have inherent activity, leading to its own activation (i.e. conversion to the disulphide-linked-two-chain form by cleavage after Thr-Lys-Gln-Ala-Arg614). Also, there is growing evidence that the activation of zymogen occurs at the cell surface and in relation to the acidification and lowering of ionic strength within cell-surface microenvironments. The present study aimed to provide evidence for the involvement of zymogen activity in its activation in physiologically relevant cellular contexts. For this purpose, the activity of a pseudozymogen form of recombinant matriptase (HL-matriptase zymogen) was examined using acetyl-l-Lys-l-Thr-l-Lys-l-Gln-l-Leu-l-Arg-4-methyl-coumaryl-7-amide as a substrate. HL-matriptase zymogen exhibited optimal activity toward the substrate pH approximately 6.0. The substrate hydrolysis at the pH value was hardly detected when NaCl was present at a concentration of 145 mM. In a buffer of pH 6.0 containing 5 mM NaCl, the activity of HL-matriptase zymogen was only approximately 30-times lower than that of the respective two-chain form. These findings suggest that the in vivo activation of matriptase zymogen occurs via a mechanism involving the zymogen activity.

This review provides some aspects on the physiology of stimulation and inhibition of pancreatic digestive enzyme secretion and the pathophysiology of pancreatic acinar cell function leading to pancreatitis. Cholecystokinin (CCK) stimulates both directly via CCK-A receptors on acinar cells and indirectly via CCK-B receptors on nerves, followed by acetylcholine release, pancreatic enzyme secretion. It is still not known whether CCK-A receptors exist in human acinar cells, in contrast to acinar cells of rodents where CCK-A receptors have been well described. CCK has numerous actions both in the periphery and in the central nervous systems. CCK inhibits gastric motility and regulates satiety. Another major function of CCK is stimulation of gallbladder contraction. This function enables that bile acids act simultaneously with pancreatic lipolytic enzymes. Secretin is a major stimulator of bicarbonate secretion. Trypsinogen is activated by the gut mucosal enzyme enterokinase. The other pancreatic proenzymes are activated by trypsin. Termination of enzyme secretion may be regulated by negative feedback mechanisms via destruction of CCK-releasing peptides by trypsin. Furthermore, the ileum may act as a brake by release of inhibitory hormones such as PYY and somatostatin. In the pathophysiology of acute pancreatitis, fusion of zymogen granules with lysosomes leading to intracellular activation of trypsinogen is regarded as an initiation step. This activation of trypsinogen may be caused by the lysosomal enzyme cathepsin B. However, autoactivation of trypsinogen itself may be a possibility in pathogenesis. Autoactivation is enhanced in certain mutations of trypsinogen. Furthermore, an imbalance of protease inhibitors and active proteases may be involved. The role of pancreatic lipolytic enzymes, the role of bicarbonate secretion, and toxic Ca2+ signals by excessive liberation from the endoplasmic reticulum have to be discussed in the pathogenesis of acute pancreatitis.

Glucagon-like peptide-1 (GLP-1) is attracting increasing interest on account of its prominent benefits in type 2 diabetes. However, its clinical application is limited because of short biological half-life. This study was designed to produce a C-terminal site-specific PEGylated analog of cysteine-mutated GLP-1 (cGLP-1) to prolong its action. The gene of cGLP-1 was inserted into pET32a to construct a thioredoxinA fusion protein. After expression in BL21 (DE3) strain, the fusion protein was purified with Ni-affinity chromatography and then was PEGylated with methoxy-polyethylene glycol-maleimide (mPEG(10K)-MAL). The PEGylated fusion protein was purified with anion exchange chromatography and then was cleaved by enterokinase. The digested product was further purified with reverse-phase chromatography. Finally, 8.7 mg mPEG(10K)-cGLP-1 with a purity of up to 98% was obtained from the original 500 ml culture. The circular dichroism spectra indicated that mPEG(10K)-cGLP-1 maintained the secondary structure of native GLP-1. As compared with that of native GLP-1, the plasma glucose lowering activity of mPEG(10K)-cGLP-1 was significantly extended. These results suggest that our method will be useful in obtaining a large quantity of mPEG(10K)-cGLP-1 for further study and mPEG(10K)-cGLP-1 might find a role in the therapy of type 2 diabetes through C-terminal site-specific PEGylation.

Complement receptor type 1 (CR1) is a versatile inhibitor of both classical and alternative pathway C3 and C5 convertases with an ability to accelerate decay activity and act as a co-factor in C3b/C4b cleavage. In order to develop a short form of CR1 with similar biological activities to the full-length human CR1, we combined functional domain 1, located in the long homologous repeat (LHR) A, with functional domain 2, located in LHR C. We expressed the two-domain, two-function protein with an enterokinase site at the N-terminus and a termination codon at the C-terminus in Escherichia coli. The fusion protein was purified on a Ni–NTA–agarose column. After subsequent refolding, the recombinant CR1-derivative protein was obtained by enterokinase cleavage and subsequent purification. In vitro, the recombinant CR1-derivative reduced hemolysis, C5a release and surface C3 deposition. It was also effective in prolonging survival of transfused incompatible red blood cells in vivo. Our results indicate that the CR1-based protein may be a model for developing smaller and more potent complement inhibitors for future therapeutics.

To develop radioimmunoassays (RIAs) for rat trypsinogens 1 and 2 and to investigate the effect of nicotine exposure on concentration and production of pancreatic zymogens in the rat. METHODS: Male Sprague-Dawley rats were supplied with either normal or nicotine-containing (0.77 mM) water for 28 days and were then killed. Rabbit antibodies for the activation peptides of trypsinogens 1 and 2 were obtained for use in the RIAs. Concentrations of the both trypsinogens in pancreatic extracts were measured by the RIAs after activation by enterokinase. DNA and amylase were measured using commercial kits. mRNA for trypsinogens 1 and 2, procolipase, and cholecystokinin receptor was measured by in situ hybridization. RESULTS: The specificity of the RIA for the trypsinogen 1 activation peptide was satisfactory. The RIA for the trypsinogen 2 activation peptide showed a limited cross-reaction toward the synthetic trypsinogen 1 activation peptide, but the importance of this cross-reaction was moderate when investigated in samples of activated trypsinogens. Weight gain was reduced in nicotine-treated animals. Concentrations of amylase, trypsinogen 1, trypsinogen 2, and the ratio of trypsinogen 2 to 1 were all increased in pancreatic extracts of nicotine-fed animals. Total DNA and mRNA for the trypsinogens, procolipase, and cholecystokinin receptor were not affected by nicotine exposure. CONCLUSIONS: The combination of increased proenzyme concentrations and unaffected mRNA levels suggests that nicotine impairs secretion rather than production of pancreatic zymogens. (Less)

We previously reported a strategy for expression and purification of human Vasostatin120-180 (VAS), a potent angiogenesis inhibitor in a GST fusion form; however, the yield of 7.2 mg per liter of culture was relatively low. The aim of this study was to develop a more efficient system to improve and facilitate the production of VAS protein in a soluble and native form in Escherichia coli. The VAS gene with optimized condons was cloned into pET28a and overexpressed as a N-terminal His-tagged fusion protein. Between His-tag and VAS, an enterokinase recognition site was introduced to release the intact VAS. Optimal expression of soluble His-VAS was achieved by examining the contribution of chaperone coexpression and lower temperature fermentation. Ammonium sulfate precipitation was first employed to remove nucleic acid and partial host proteins. When further purified by Ni 2+ affinity chromatography, 40 mg of His-VAS was isolated with purity over 85% from 1 L of culture. After desalting with Sephadex G15 and digestion with His-EK, followed by the removal of the His-tag and His-EK with Ni 2+ -NTA resin, 21 mg of intact VAS was finally obtained from 1 L of bacterial culture, which was approximately 3-fold the yield we previously obtained via GST fusion expression strategy. The identity of His-VAS and VAS was confirmed by Western blot. Purified VAS displayed distinct anti-angiogenic activity, which was shown by the endothelial cell proliferation inhibition assay and chicken chorioallantoic membrane assay. In sum, we greatly improved the yield of intact and bioactive VAS protein, and using this successful example, we propose a more efficient system for the high-level production of intact functional proteins, especially for low molecule weight peptides.

Background A noninvasive model of necrohemorrhagic pancreatitis induced by simultaneous intravenous cerulein/enterokinase (EK) infusion has recently been established in rats. The aim of the present study was to establish this new model in mice and to compare it with the rat model. Methods Male Balb/C mice (20 to 25 g) were used for the experiments. Pancreatitis was induced by simultaneous intravenous infusion of cerulein and EK. Controls were infused with either 0.9% NaCl, cerulein, or EK. Animals were humanely killed 6 hours after start of infusions. Pancreatic and pulmonary injury was assessed by histology, wet-to-dry weight ratio, and myeloperoxidase activity. Systemic cytokine, amylase, and lactate dehydrogenase (LDH) levels in blood were measured to assess pancreatic and systemic inflammatory response. To evaluate the role of protease activity in this model, trypsin, cathepsin B, and elastase activity were measured in pancreatic tissue. Survival experiments were performed to determine survival time and tissue injury in the later course of the disease. Results Mice with simultaneous cerulein/EK infusion developed marked local and systemic organ injury compared with those animals who received cerulein or EK alone. Pancreatic and pulmonary injury increased with high concentrations of cerulein/EK infusions. Survival decreased in these animals. Whereas acinar cell apoptosis was an early finding, pancreatic necrosis was observed later in the course of the disease. Serum levels of LDH, interleukin (IL)-1α, and IL-1β reflected cell damage and the systemic inflammatory response. Protease activity in pancreatic tissue was greatest in animals with simultaneous cerulein/EK infusion. Conclusions Using intravenous cerulein/EK infusions, a model of lethal acute pancreatitis has been established in mice. Major pancreatic edema, acinar cell apoptosis and necrosis, and pulmonary leukocyte sequestration are characteristic findings in this model. Although pancreatic injury was not as strong as in the rat model, this model may prove useful for future studies in transgenic mice.

OBJECTIVES The aim of the present study was to purify and clone the trypsinogen isoforms from the guinea pig pancreas and characterize their activation properties. METHODS Trypsinogens from pancreatic homogenates were isolated by ecotin-affinity chromatography, followed by cation-exchange chromatography. Activation of trypsinogens was tested with enteropeptidase, cathepsin B, and trypsin. Complementary DNAs for pretrypsinogens were cloned from total RNA after reverse transcription and polymerase chain reaction amplification. RESULTS Purification of trypsinogens yielded a single peak with an N-terminal amino-acid sequence of LPIDD. Cloning of pretrypsinogen cDNAs revealed 2 distinct but nearly identical isoforms. At the amino acid level, the only difference between the 2 isoforms is an Ala/Ser change at position 15 within the signal peptide. Thus, both cDNA variants give rise to the same mature trypsinogen upon secretion. Guinea pig trypsinogen is readily activated by enteropeptidase and cathepsin B but exhibits essentially no autoactivation, under conditions where human cationic and anionic trypsinogens rapidly autoactivate. CONCLUSIONS The observations suggest that multiple trypsinogen isoforms and their ability to autoactivate are not required universally for normal digestive physiology in mammals. Furthermore, the inability of guinea pig trypsinogen to undergo autoactivation suggests that this species might be more resistant to pancreatitis than humans, where increased autoactivation of cationic trypsinogen mutants has been linked to hereditary pancreatitis.

TTSPs [type II TMPRSSs (transmembrane serine proteases)] are a growing family of trypsin-like enzymes with, in some cases, restricted tissue distribution. To investigate the expression of TTSPs in the nervous system, we performed a PCR-based screening approach with P10 (postnatal day 10) mouse spinal cord mRNA. We detected the expression of five known TTSPs and identified a novel TTSP, which we designated neurobin. Neurobin consists of 431 amino acids. In the extracellular part, neurobin contains a single SEA (sea-urchin sperm protein, enterokinase and agrin) domain and a C-terminal serine protease domain. RT–PCR (reverse transcription–PCR) analysis indicated the expression of neurobin in spinal cord and cerebellum. Histochemical analysis of brain sections revealed distinct staining of Purkinje neurons of the cerebellum. Transiently overexpressed neurobin was autocatalytically processed and inserted into the plasma membrane. Autocatalytic activation could be suppressed by mutating Ser381 in the catalytic pocket to an alanine residue. The protease domain of neurobin, produced in Escherichia coli and refolded from inclusion bodies, cleaved chromogenic peptides with an arginine residue in position P1. Serine protease inhibitors effectively suppressed the proteolytic activity of recombinant neurobin. Ca2+ or Na+ ions did not significantly modulate the catalytic activity of the protease. Recombinant neurobin processed 17-kDa FGF-2 (fibroblast growth factor-2) at several P1 lysine and arginine positions to distinct fragments, in a heparin-inhibitable manner, but did not cleave FGF-7, laminin or fibronectin. These results indicate that neurobin is an authentic TTSP with trypsin-like activity and is able to process FGF-2 in vitro.

Mytilus californianus foot protein three (Mcfp-3) was successfully expressed in the yeast, Kluyveromyces lactis . The first nine amino acids (YPYDVPDYA) from the human-influenza-virus hemagglutinin (HA) protein were fused to the amino terminus of Mcfp-3 (HA–Mcfp-3) to facilitate identification and purification. HA–Mcfp-3 was purified to a concentration of 1 mg/L using HA affinity chromatography. The recovered polypeptide was resolved by SDS–PAGE and migrated primarily at 36 kDa, an increase of approximately 29 kDa over the calculated molecular weight of a HA–Mcfp-3 monomer. Significantly, release of Mcfp-3 by enterokinase treatment coincided with the formation of high molecular weight complexes. It is noteworthy that the complexes mimicked the previously reported insolubility of Mcfps found in vivo to denaturing and reducing conditions. These data demonstrate the successful expression of Mcfp-3 in K. lactis and show an intrinsic ability of Mcfp-3 to self-assemble into stable, higher molecular weight forms.

Exendin-4, a peptide analogue of glucagon-like peptide-1 (GLP-1), has been developed for treatment of type 2 diabetes. Herein, the secretive exendin-4 fusion protein, expressed by methanol induction in Pichia pastoris system, was purified to homogeneity by chromatography followed by enterokinase cleavage of the fusion protein and subsequent purification of the recombinant exendin-4. Purity of the recombinant exendin-4 was 95.6%. Bioactivity assay revealed that it had glucose-lowering and insulin-releasing action in vivo.

Although principally produced by the pancreas to degrade dietary proteins in the intestine, trypsins are also expressed in the nervous system and in epithelial tissues, where they have diverse actions that could be mediated by protease-activated receptors (PARs). We examined the biological actions of human trypsin IV (or mesotrypsin) and rat p23, inhibitor-resistant forms of trypsin. The zymogens trypsinogen IV and pro-p23 were expressed in Escherichia coli and purified to apparent homogeneity. Enteropeptidase cleaved both zymogens, liberating active trypsin IV and p23, which were resistant to soybean trypsin inhibitor and aprotinin. Trypsin IV cleaved N-terminal fragments of PAR(1), PAR(2), and PAR(4) at sites that would expose the tethered ligand (PAR(1) = PAR(4) > PAR(2)). Trypsin IV increased [Ca(2+)](i) in transfected cells expressing human PAR(1) and PAR(2) with similar potencies (PAR(1), 0.5 microm; PAR(2), 0.6 microm). p23 also cleaved fragments of PAR(1) and PAR(2) and signaled to cells expressing these receptors. Trypsin IV and p23 increased [Ca(2+)](i) in rat dorsal root ganglion neurons that responded to capsaicin and which thus mediate neurogenic inflammation and nociception. Intraplantar injection of trypsin IV and p23 in mice induced edema and granulocyte infiltration, which were not observed in PAR (-/-)(1)(trypsin IV) and PAR (-/-)(2) (trypsin IV and p23) mice. Trypsin IV and p23 caused thermal hyperalgesia and mechanical allodynia and hyperalgesia in mice, and these effects were absent in PAR (-/-)(2) mice but maintained in PAR (-/-)(1) mice. Thus, trypsin IV and p23 are inhibitor-resistant trypsins that can cleave and activate PARs, causing PAR(1)- and PAR(2)-dependent inflammation and PAR(2)-dependent hyperalgesia.