Characterization and application of natural and recombinant butelase-1 to improve industrial enzymes by end-to-end circularization.

Butelase-1, an asparaginyl endopeptidase or legumain, is the prototypical and fastest known Asn/Asp-specific peptide ligase. It is highly useful for engineering and macrocyclization of peptides and proteins. However, certain biochemical properties and applications of naturally occurring and recombinant butelase-1 remain unexplored. Here we report methods to increase the yield of natural and bacterial expressed recombinant butelase-1 and how they can be used to improve the stability and activity of two important industrial enzymes, lipase and phytase, by end-to-end circularization. First, the yield of natural butelase-1 was increased 3-fold to 15 mg kg-1 by determining its highest distribution which is found in young tissues, such as shoots. The yield of recombinantly-produced soluble butelase-1 was improved by promoting cytoplasmic disulfide folding, codon changes, and truncation of the N-terminal pro-domain. Natural and recombinant butelase-1 displayed similar ligase activity, physical stability, and salt tolerance. Furthermore, the processing and glycosylation sites of natural and recombinant butelase-1 were determined by proteomic analysis. Storage conditions for both forms of butelase-1, frozen or lyophilized, were also optimized. Cyclization of lipase and phytase mediated by either soluble or immobilized butelase-1 was highly efficient and simple, and resulted in increased thermal stability and enhanced enzymatic activity. Overall, improved production of butelase-1 can be exploited to improve the biocatalytic efficacy of lipase and phytase by end-to-end cyclization. In turn, ligase-improved enzymes could be a general and environmentally friendly strategy for producing more stable and efficient industrial enzymes.

Cyclization of a G4-specific peptide enhances its stability and G-quadruplex binding affinity.

G-quadruplexes (G4) are non-canonical nucleic acid structures with important implications in biology. Based on an α-helical fragment of the RHAU helicase that displays high specificity for parallel-stranded G-quadrplexes, herein we demonstrate its head-to-tail cyclization by a high-efficiency ligase. The cyclic peptide exhibits superior stability and binding affinity to a G-quadruplex, and can serve as an excellent investigational tool for chemical biology applications.

Ginsentides: Cysteine and Glycine-rich Peptides from the Ginseng Family with Unusual Disulfide Connectivity.

Ginseng, a popular and valuable traditional medicine, has been used for centuries to maintain health and treat disease. Here we report the discovery and characterization of ginsentides, a novel family of cysteine and glycine-rich peptides derived from the three most widely-used ginseng species: Panax ginseng, Panax quinquefolius, and Panax notoginseng. Using proteomic and transcriptomic methods, we identified 14 ginsentides, TP1-TP14 which consist of 31-33 amino acids and whose expression profiles are species- and tissues-dependent. Ginsentides have an eight-cysteine motif typical of the eight-cysteine-hevein-like peptides (8C-HLP) commonly found in medicinal herbs, but lack a chitin-binding domain. Transcriptomic analysis showed that the three-domain biosynthetic precursors of ginsentides differ from known 8C-HLP precursors in architecture and the absence of a C-terminal protein-cargo domain. A database search revealed an additional 50 ginsentide-like precursors from both gymnosperms and angiosperms. Disulfide mapping and structure determination of the ginsentide TP1 revealed a novel disulfide connectivity that differs from the 8C-HLPs. The structure of ginsentide TP1 is highly compact, with the N- and C-termini topologically fixed by disulfide bonds to form a pseudocyclic structure that confers resistance to heat, proteolysis, and acid and serum-mediated degradation. Together, our results expand the chemical space of natural products found in ginseng and highlight the occurrence, distribution, disulfide connectivity, and precursor architectures of cysteine- and glycine-rich ginsentides as a class of novel non-chitin-binding, non-cargo-carrying 8C-HLPs.

Trends in mortality risks among 94,328 patients surviving 30days after a first isolated coronary artery bypass graft procedure from 1987 to 2006: A population-based study.

BACKGROUND: Updated knowledge about survival after coronary artery bypass graft (CABG) surgery is needed. We examined 20-year trends in 4-year survival after a first isolated CABG procedure, compared with that of the general population. METHODS: We identified 94,328 patients surviving 30days after a first isolated CABG 1987-2006 from the Swedish Inpatient Register. RESULTS: Crude annual mortality rates remained stable at approximately 1% in patients aged 18-54years and at approximately 2% in those aged ≥55years. After adjustment for comorbidities, 4-year survival in men aged 18-54 and ≥55years improved by 37% (HR: 0.63, 95% CI, 0.46-0.88) and 31% (HR: 0.69, 95% CI, 0.63-0.76), respectively, (1987-1991 vs. 2002-2006). The corresponding estimate for women aged ≥55years was 38% (HR: 0.62, 95% CI, 0.52-0.75), with no significant change in survival in women aged <55years (HR: 1.02, 95% CI, 0.52-2.03). Men and women aged <55years had higher mortality than the general population, with standardized mortality ratios (SMR) of 1.76 (95% CI, 1.35-2.22) in men and 4.49 (95% CI, 2.74-6.68) in women during the last period (2002-2006). In contrast, patients aged ≥55years had better survival with a SMR of 0.74 (95% CI, 0.70-0.78) in men and 0.82 (95% CI, 0.74-0.91) in women during 2002-2006. CONCLUSION: During 1987-2006, there was a significant improvement in survival after CABG for all categories, except in women aged <55years. Men and women aged ≥55years who survived the first 30days after CABG had a lower mortality risk than the general population.

Costs of providing tuberculosis diagnosis and treatment services in Viet Nam.

OBJECTIVE: To estimate the cost of providing tuberculosis (TB) diagnosis and treatment packages at different levels of health facilities in Viet Nam. DESIGN: This was a retrospective costing study from the providers' perspective using a standard costing approach. We included typical services for TB diagnosis and treatment based on standard protocols. RESULTS: The least expensive TB service was the 6-month isoniazid preventive therapy regimen for latent tuberculous infection provided by district health centres (US$7.20-14.30, accounting for 0.3-0.7% of Viet Nam's per capita gross domestic product [GDP] of US$2052.30 in 2014). The cost of diagnosing and treating a patient with drug-susceptible TB (the most common type of TB) ranged between US$51.20 and US$180.70, and represented 2.5-8.8% of Viet Nam's per capita GDP in 2014. The most expensive TB service was the diagnosis and treatment of a multidrug-resistant TB case (US$1568.20-2391.20), accounting for 76.4-116.5% of Viet Nam's per capita GDP in 2014). CONCLUSION: The cost of TB diagnosis and treatment services in Viet Nam varied according to level of health facility, type of TB, different costing options, and different staff cost scenarios.

Enzymatic Engineering of Live Bacterial Cell Surfaces Using Butelase†1.

Butelase-mediated ligation (BML) can be used to modify live bacterial cell surfaces with diverse cargo molecules. Surface-displayed butelase recognition motif NHV was first introduced at the C-terminal end of the anchoring protein OmpA on E. coli cells. This then served as a handle of BML for the functionalization of E. coli cell surfaces with fluorescein and biotin tags, a tumor-associated monoglycosylated peptide, and mCherry protein. The cell-surface ligation reaction was achieved at low concentrations of butelase and the labeling substrates. Furthermore, the fluorescein-labeled bacterial cells were used to show the interactions with cultured HeLa cells and with macrophages in live transgenic zebrafish, capturing the latter's powerful phagocytic effect in action. Together these results highlight the usefulness of butelase 1 in live bacterial cell surface engineering for novel applications.

Engineering a Catalytically Efficient Recombinant Protein Ligase.

Breaking and forming peptidyl bonds are fundamental biochemical reactions in protein chemistry. Unlike proteases that are abundantly available, fast-acting ligases are rare. OaAEP1 is an enzyme isolated from the cyclotide-producing plant oldenlandia affinis that displayed weak peptide cyclase activity, despite having a similar structural fold with other asparaginyl endopeptidases (AEP). Here we report the first atomic structure of OaAEP1, at a resolution of 2.56 Å, in its preactivation form. Our structure and biochemical analysis of this enzyme reveals its activation mechanism as well as structural features important for its ligation activity. Importantly, through structure-based mutagenesis of OaAEP1, we obtained an ultrafast variant having hundreds of times faster catalytic kinetics, capable of ligating well-folded protein substrates using only a submicromolar concentration of enzyme. In contrast, the protein-protein ligation activity in the original wild-type OaAEP1 enzyme described previously is extremely weak. Thus, the structure-based engineering of OaAEP1 described here provides a unique and novel recombinant tool that can now be used to conduct various protein labeling and modifications that were extremely challenging before.

Identification and Characterization of Roseltide, a Knottin-type Neutrophil Elastase Inhibitor Derived from Hibiscus sabdariffa.

Plant knottins are of therapeutic interest due to their high metabolic stability and inhibitory activity against proteinases involved in human diseases. The only knottin-type proteinase inhibitor against porcine pancreatic elastase was first identified from the squash family in 1989. Here, we report the identification and characterization of a knottin-type human neutrophil elastase inhibitor from Hibiscus sabdariffa of the Malvaceae family. Combining proteomic and transcriptomic methods, we identified a panel of novel cysteine-rich peptides, roseltides (rT1-rT8), which range from 27 to 39 residues with six conserved cysteine residues. The 27-residue roseltide rT1 contains a cysteine spacing and amino acid sequence that is different from the squash knottin-type elastase inhibitor. NMR analysis demonstrated that roseltide rT1 adopts a cystine-knot fold. Transcriptome analyses suggested that roseltides are bioprocessed by asparagine endopeptidases from a three-domain precursor. The cystine-knot structure of roseltide rT1 confers its high resistance against degradation by endopeptidases, 0.2 N HCl, and human serum. Roseltide rT1 was shown to inhibit human neutrophil elastase using enzymatic and pull-down assays. Additionally, roseltide rT1 ameliorates neutrophil elastase-stimulated cAMP accumulation in vitro. Taken together, our findings demonstrate that roseltide rT1 is a novel knottin-type neutrophil elastase inhibitor with therapeutic potential for neutrophil elastase associated diseases.

Butelase-Mediated Ligation as an Efficient Bioconjugation Method for the Synthesis of Peptide Dendrimers.

Herein we report a novel enzymatic bioconjugation method to prepare peptide dendrimers. Under the catalysis of a newly discovered peptide ligase, butelase 1, peptide dendrimers of di-, tetra-, and octabranches were successfully synthesized using thiodepsipeptides as acyl donors for ligation with lysyl dendrimeric scaffolds. The efficient assembly of the highly clustered dendrimeric structure highlighted the versatility of butelase 1. We also showed that our synthetic antibacterial peptide dendrimers containing an RLYR motif are highly potent and broadly active against antibiotic-resistant strains.

Butelase-mediated cyclization and ligation of peptides and proteins.

Enzymes that catalyze efficient macrocyclization or site-specific ligation of peptides and proteins can enable tools for drug design and protein engineering. Here we describe a protocol to use butelase 1, a recently discovered peptide ligase, for high-efficiency cyclization and ligation of peptides and proteins ranging in size from 10 to >200 residues. Butelase 1 is the fastest known ligase and is found in pods of the common medicinal plant Clitoria ternatea (also known as butterfly pea). It has a very simple C-terminal-specific recognition motif that requires Asn/Asp (Asx) at the P1 position and a dipeptide His-Val at the P1' and P2' positions. Substrates for butelase-mediated ligation can be prepared by standard Fmoc (9-fluorenylmethyloxycarbonyl) chemistry or recombinant expression with the minimal addition of this tripeptide Asn-His-Val motif at the C terminus. Butelase 1 achieves cyclizations that are 20,000 times faster than those of sortase A, a commonly used enzyme for backbone cyclization. Unlike sortase A, butelase is traceless, and it can be used for the total synthesis of naturally occurring peptides and proteins. Furthermore, butelase 1 is also useful for intermolecular ligations and synthesis of peptide or protein thioesters, which are versatile activated intermediates necessary for and compatible with many chemical ligation methods. The protocol describes steps for isolation and purification of butelase 1 from plant extract using a four-step chromatography procedure, which takes ∼3 d. We then describe steps for intramolecular cyclization, intermolecular ligation and butelase-mediated synthesis of protein thioesters. Butelase reactions are generally completed within minutes and often achieve excellent yields.

Butelase-Mediated Macrocyclization of d-Amino-Acid-Containing Peptides.

Macrocyclic compounds have received increasing attention in recent years. With their large surface area, they hold promise for inhibiting protein-protein interactions, a chemical space that was thought to be undruggable. Although many chemical methods have been developed for peptide macrocyclization, enzymatic methods have emerged as a promising new economical approach. Thus far, most enzymes have been shown to act on l-peptides; their ability to cyclize d-amino-acid-containing peptides has rarely been documented. Herein we show that macrocycles consisting of d-amino acids, except for the Asn residue at the ligating site, were efficiently synthesized by butelase 1, an Asn/Asp-specific ligase. Furthermore, by using a peptide-library approach, we show that butelase 1 tolerates most of the d-amino acid residues at the P1'' and P2'' positions.

Total Synthesis of Circular Bacteriocins by Butelase 1.

Circular bacteriocins, ranging from 35 to 70 amino acids, are the largest cyclic peptides produced by lactic acid bacteria to suppress growth of other bacteria. Their end-to-end cyclized backbone that enhances molecular stability is an advantage to survive in pasteurization and cooking processes in food preservation, but becomes a disadvantage and challenge in chemical synthesis. They also contain unusually long and highly hydrophobic segments which pose an additional synthetic challenge. Here we report the total synthesis of the three largest circular bacteriocins, AS-48, uberolysin, and garvicin ML, by an efficient chemoenzymatic strategy. A key feature of our synthetic scheme is the use of an Asn-specific butelase-mediated cyclization of their linear precursors, prepared by microwave stepwise synthesis. Antimicrobial assays showed that the AS-48 linear precursor is inactive at concentrations up to 100 µM, whereas the macrocyclic AS-48 is potently active against pathogenic and drug-resistant bacteria, with minimal inhibitory concentrations in a sub-micromolar range.

A high-throughput peptidomic strategy to decipher the molecular diversity of cyclic cysteine-rich peptides.

Cyclotides are plant cyclic cysteine-rich peptides (CRPs). The cyclic nature is reported to be gene-determined with a precursor containing a cyclization-competent domain which contains an essential C-terminal Asn/Asp (Asx) processing signal recognized by a cyclase. Linear forms of cyclotides are rare and are likely uncyclizable because they lack this essential C-terminal Asx signal (uncyclotide). Here we show that in the cyclotide-producing plant Clitoria ternatea, both cyclic and acyclic products, collectively named cliotides, can be bioprocessed from the same cyclization-competent precursor. Using an improved peptidomic strategy coupled with the novel Asx-specific endopeptidase butelase 2 to linearize cliotides at a biosynthetic ligation site for transcriptomic analysis, we characterized 272 cliotides derived from 38 genes. Several types of post-translational modifications of the processed cyclotides were observed, including deamidation, oxidation, hydroxylation, dehydration, glycosylation, methylation, and truncation. Taken together, our results suggest that cyclotide biosynthesis involves 'fuzzy' processing of precursors into both cyclic and linear forms as well as post-translational modifications to achieve molecular diversity, which is a commonly found trait of natural product biosynthesis.

Butelase 1: A Versatile Ligase for Peptide and Protein Macrocyclization.

Macrocyclization is a valuable tool for drug design and protein engineering. Although various methods have been developed to prepare macrocycles, a general and efficient strategy is needed. Here we report a highly efficient method using butelase 1 to macrocyclize peptides and proteins ranging in sizes from 26 to >200 residues. We achieved cyclizations that are 20,000 times faster than sortase A, the most widely used ligase for protein cyclization. The reactions completed within minutes with up to 95% yields.

Site-Specific N-Terminal Labeling of Peptides and Proteins using Butelase†1 and Thiodepsipeptide.

An efficient ligase with exquisite site-specificity is highly desirable for protein modification. Recently, we discovered the fastest known ligase called butelase 1 from Clitoria ternatea for intramolecular cyclization. For intermolecular ligation, butelase 1 requires an excess amount of a substrate to suppress the reverse reaction, a feature similar to other ligases. Herein, we describe the use of thiodepsipeptide substrates with a thiol as a leaving group and an unacceptable nucleophile to render the butelase-mediated ligation reactions irreversible and in high yields. Butelase 1 also accepted depsipeptides as substrates, but unlike a thiodesipeptide, the desipeptide ligation was partially reversible as butelase 1 can tolerate an alcohol group as a poor nucleophile. The thiodesipeptide method was successfully applied in N-terminal labeling of ubiquitin and green fluorescent protein using substrates with or without a biotin group in high yields.

Butelase-mediated synthesis of protein thioesters and its application for tandem chemoenzymatic ligation.

Using a recently discovered peptide ligase, butelase 1, we developed a novel method to access protein thioesters in good yield. We successfully combined it with native chemical ligation and sortase-mediated ligation in tandem for protein C-terminal labeling and dual-terminal labeling to exploit the orthogonality of these three ligation methods.

Allotides: Proline-Rich Cystine Knot α-Amylase Inhibitors from Allamanda cathartica.

Cystine knot α-amylase inhibitors belong to a knottin family of peptidyl inhibitors of 30-32 residues and contain two to four prolines. Thus far, only four members of the group of cystine knot α-amylase inhibitors have been characterized. Herein, the discovery and characterization of five cystine knot α-amylase inhibitors, allotides C1-C5 (Ac1-Ac5) (1-5), from the medicinal plant Allamanda cathartica are reported using both proteomic and genomic methods. Proteomic analysis showed that 1-5 are 30 amino acids in length with three or four proline residues. NMR determination of 4 revealed that it has two cis- and one trans-proline residues and adopts two equally populated conformations in solution. Determination of disulfide connectivity of 2 by differential S-reduction and S-alkylation provided clues of its unfolding process. Genomic analysis showed that allotide precursors contain a three-domain arrangement commonly found in plant cystine knot peptides with conserved residues flanking the processing sites of the mature allotide domain. This work expands the number of known cystine knot α-amylase inhibitors and furthers the understanding of both the structural and biological diversity of this type of knottin family.

Antimicrobial light-activated materials: towards application for food and environmental safety.

AIMS: To produce light-activated antimicrobial materials composed of the photodynamic dye phloxine B incorporated into paper or cellulose membranes and to investigate ability of these materials to decrease bacterial loads on their surfaces as well as on food surfaces that were in contact with these materials under illumination with regular white light. METHODS AND RESULTS: Antimicrobial cellulose-based materials with incorporated phloxine B were produced using a layer-by-layer deposition method. Antimicrobial properties of the materials were tested in model systems as well as for decontamination of food and food contact surfaces. Pseudomonas aeruginosa, Listeria monocytogenes and Bacillus anthracis were efficiently killed by exposure of the bacterial suspension to the dye-containing material under illumination with white light, but Salmonella Typhimurium and Escherichia coli O157:H7 were only partially affected. Application of the materials for decontamination of food surfaces artificially contaminated with L. monocytogenes was shown to be ineffective, while the self-decontamination of the material surface by exposure to white light resulted in eradication of L. monocytogenes cells from the material surface. CONCLUSIONS: The developed materials showed significant self-decontaminating ability when under illumination; however, decontamination of food surfaces in contact with the developed materials was not achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates the antibacterial activity of materials with incorporated photodynamic dyes when under illumination with regular-intensity white light. Possible uses of the light-activated antimicrobial materials for food processing, as food contact surfaces, and surfaces in public areas to prevent cross-contamination are discussed.

Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis.

Proteases are ubiquitous in nature, whereas naturally occurring peptide ligases, enzymes catalyzing the reverse reactions of proteases, are rare occurrences. Here we describe the discovery of butelase 1, to our knowledge the first asparagine/aspartate (Asx) peptide ligase to be reported. This highly efficient enzyme was isolated from Clitoria ternatea, a cyclic peptide-producing medicinal plant. Butelase 1 shares 71% sequence identity and the same catalytic triad with legumain proteases but does not hydrolyze the protease substrate of legumain. Instead, butelase 1 cyclizes various peptides of plant and animal origin with yields greater than 95%. With Kcat values of up to 17 s(-1) and catalytic efficiencies as high as 542,000 M(-1) s(-1), butelase 1 is the fastest peptide ligase known. Notably, butelase 1 also displays broad specificity for the N-terminal amino acids of the peptide substrate, thus providing a new tool for C terminus-specific intermolecular peptide ligations.

Sex-specific trends in 4-year survival in 37 276 men and women with acute myocardial infarction before the age of 55 years in Sweden, 1987-2006: a register-based cohort study.

OBJECTIVE: To examine sex-specific trends in 4-year mortality among young patients with first acute myocardial infarction (AMI), 1987-2006. DESIGN: Prospective cohort study. SETTING: Sweden. PARTICIPANTS: We identified 37 276 cases (19.4% women; age, 25-54 years) from the Swedish Inpatient Register, 1987-2006, who had survived 28 days after an AMI. OUTCOME MEASURES: 4-year mortality from all causes and standard mortality ratio (SMR). RESULTS: From the first to last 5-year period, the absolute excess risk decreased from 1.38 to 0.50 and 1.53 to 0.59 per 100 person-years among men aged 25-44 and 45-54 years, respectively. Corresponding figures for women were a decrease from 2.26 to 1.17 and from 1.93 to 1.45 per 100 person-years, respectively. Trends for women were non-linear, decreasing to the same extent as those for men until the third period, then increasing. For the last 5-year period, the standardised mortality ratio for young survivors of AMI compared with the general population was 4.34 (95% CI 3.04 to 5.87) and 2.43 (95% CI 2.12 to 2.76) for men aged 25-44 and 45-54 years, respectively, and 13.53 (95% CI 8.36 to 19.93) and 6.42 (95% CI 5.24 to 7.73) for women, respectively. Deaths not associated with cardiovascular causes increased from 21.5% to 44.6% in men and 41.5% to 65.9% in women. CONCLUSIONS: Young male survivors of AMI have low absolute long-term mortality rates, but these rates remain twofold to fourfold that of the general population. After favourable development until 2001, women now have higher absolute mortality than men and a 6-fold to 14-fold risk of death compared with women in the general population.