Assembly and selective "in synthesis" labeling of quenched fluorogenic protease substrates

Because impaired cellular protease activities are linked to many diseases, such as cancer, inflammation, neurodegeneration, and infection, internally quenched fluorescent peptides have recently been developed as tools for analyzing the specificities of these enzymes. Here we report convenient and cost-effective approaches for the selective "in synthesis" assembly of such substrate peptides for protease assays. Fluorescein and Dabcyl groups were covalently and selectively attached during synthesis to epsilon-amino groups of internal lysines. Functionality was then tested by digestion with leucine aminopeptidase, chymotrypsin, and microsomal vesicles. All peptides proved to be appropriate substrates of the enzymes tested and of the endogenous peptidases in the microsomal vesicles. In summary, we describe an innovative and cheap method to develop completely functional quenched fluorescent peptides that are usable in specific detection of individual proteases, in particular aminopeptidases, in both in vitro and in vivo systems.     

Reversible activation of cellular factor XIII by calcium

Factor XIII (FXIII) is a pro-transglutaminase found in the plasma as well as intracellularly in platelets and macrophages. Plasma FXIII is activated by thrombin cleavage (FXIIIa*) and acts in the final stages of blood coagulation cascade. In contrast, the function and activation of cellular FXIII are less characterized. Cellular FXIII relies on a conformational activation of the protein. The nonproteolytic activation of FXIII to FXIIIa° induced by Ca(2+) alone is well known, but up until now it has been discussed under which conditions the process can be induced and whether it can be reversed. Here, we study the nature of the Ca(2+)-induced FXIII activation. Previously used methods to evaluate FXIII activity detect both FXIIIa* and FXIIIa° because they rely on occurrence of enzyme activity or on active site Cys-314 solvent accessibility. Therefore, an analytical HPLC method was developed that separates zymogen recombinant FXIII (rFXIII) from rFXIIIa°. The data demonstrate that nonproteolytic activation and deactivation are highly dependent on Ca(2+) concentration, buffer, and salt components. Moreover, it is established that Ca(2+) activation of rFXIII is fully reversible, and only 2-5 mm CaCl(2) is sufficient to retain full rFXIIIa° activity. However, below 2 mm CaCl(2) the rFXIIIa° molecule deactivates. The deactivated molecule can subsequently undergo a new activation round. Furthermore, it is demonstrated that thermal stress of freeze-dried rFXIII can induce a new predisposed form that activates faster than nonstressed rFXIII.     

Comparative characterization of direct and indirect substrate probes for on-chip transamidating activity assay of transglutaminases

The development of molecular probes is a prerequisite for activity-based protein profiling. This strategy helps in characterizing the catalytic activity and function of proteins, and how these proteins and protein complexes control biological processes of interest. These probes are composed of a reactive functional group and a reporter tag. The reactive group of these substrate probes has been considered to be important to their design, while the significance of the reporter tag is relatively underestimated. In this study we compare TAMRA-cadaverine and biotin-cadaverine, two substrate probes that have different reporter tags but an identical reactive functional group. We assess the on-chip transamidating activity of two transglutaminases; transglutaminase 2 and blood coagulation factor XIII. Activity assays were more easily executed when using the direct probe TAMRA-cadaverine. However the indirect probe, biotin-cadaverine, provided a wider dynamic range, higher signal-to-noise ratio, and lower limit of detection compared to TAMRA-cadaverine. Additionally, we successfully used the on-chip activity assay using the indirect probe to determine TG2 and FXIII activities in Hela cell lysates and human plasma samples, respectively. These results demonstrate that the reporter tag of the substrate probe is critical for protocol execution, sensitivity, and dynamic range of enzyme activity assays. Furthermore, this study provides a helpful guide for development of new probes, which is necessary for the identification of potential biomarkers and therapeutic targets for treating enzyme-related diseases.     

Synthesis of substrates for periodate-coupled assay of phospholipases C and sphingomyelinases

A series of 4-nitrophenyl (pNP) and 4-methylumbelliferyl (4MU) substrate analogues of phosphatidyl choline (PC) and phosphatidic acid (PA) were synthesized from 4-bromo-1-butene by ether formation, olefin epoxidation and ring opening with the phosphate head group. The pNP PC analogue, 4-(4-nitrophenoxy)-2-hydroxy-butyl-1-phosphoryl choline (1) was evaluated in assays of fungal sphingomyelinases, also displaying phospholipase C activity. Reactions were terminated with a periodate-containing stop solution, leading to liberation of pNP, quantified spectrophotometrically in an end-point measurement. A kinetic evaluation of sphingomyelinases from Kionochaeta sp. and Penicillium emersonii showed relatively high K_M and low k_cat values for this substrate, limiting its practical applicability in assays with low sphingomyelinase concentrations.     

A highly sensitive fluorometric assay for determination of human coagulation factor XIII in plasma

Based on the iso-peptidase activity of human plasma FXIII, a novel fluorometric assay that determines FXIII concentrations in human plasma below 0.05 IU/ml is introduced. We considered a peptide sequence derived from alpha(2)-antiplasmin (n =12) to yield high sensitivity. Peptide Abz-NE(Cad-Dnp)EQVSPLTLLK exhibits a K(m) value of 19.8+/-2.8 microM and is used in a concentration of 50 microM. The assay design is suitable for measurements in cuvettes (1 ml volume) as well as for the microtiter plate (MTP) format (0.2 ml volume). It provides linear dose-response curves over a wide range of FXIII concentrations (0.05-8.8 IU/ml). The assay was validated with respect to precision, detection and quantitation limits, accuracy/specificity, linearity, and range. A comparison of the fluorometric assay with the photometric assay for FXIII determinations in plasma pools as well as single donor plasma revealed suitability of the fluorometric assay for FXIII determination in plasma of healthy individuals. FXIII concentrations in plasma samples of patients with severe FXIII deficiency are discussed in the context of FXIII antigen levels. These assays correlate well in the critical range below 0.1 IU/ml, whereas the photometric assay may overestimate residual FXIII activity in severe FXIII-deficient patients.     

Synthesis and evaluation of novel fluorogenic substrates for the detection of bacterial beta-galactosidase

AIMS: A widely used coumarin derivative is 7-hydroxy-4-methylcoumarin-beta-D-galactoside (4-methylumbelliferone-beta-D-galactoside; 4-MU-GAL). This galactoside is utilized as a substrate for the detection of the beta-galactosidase activity of coliform bacteria in water analysis. The intense fluorescence of coumarin-based molecules has enabled them to be incorporated into enzyme-based tests for the quantitative assay of indicator bacteria. The aim of this present study was to evaluate the potential of other coumarin derivatives, by synthesis of a selection of core coumarin molecules. METHODS AND RESULTS: Several coumarin derivatives were found to be more promising than 4-MU, with ethyl-7-hydroxycoumarin-3-carboxylate (EHC) giving a combination of greater fluorescence over a broad pH range and reduced growth inhibition with 12 representative coliform strains. On conversion to a beta-galactoside derivative, EHC-GAL generated a more rapid fluorescence than any other tested substrate. CONCLUSIONS: When tested in a broth assay format, based on most probable number (MPN), low numbers of coliforms were detected with EHC-GAL around 1 h earlier than with 4-MU-GAL. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study suggests that EHC-GAL should be evaluated as a substrate for the detection of coliforms in water analysis, due to a combination of the following favourable features: (i) reduced toxicity; (ii) increased fluorescence; (iii) pH stability of fluorescence; and (iv) rapid detection.     

Synthesis of tripeptide RGD amide by a combination of chemical and enzymatic methods

The tripeptide Bz-Arg-Gly-Asp(NH₂)OH was synthesized by a combination of chemical and enzymatic methods in this study. Firstly, Gly-Asp-(NH₂)₂ was synthesized by a novel chemical method in three steps including chloroacetylation of l-aspartic acid, esterification of chloroacetyl l-aspartic acid and ammonolysis of chloroacetyl l-aspartic acid diethyl ester. Secondly, the linkage of the third amino acid (Bz-Arg-OEt) to Gly-Asp-(NH₂)₂ was completed by enzymatic method under kinetic control condition. An industrial alkaline protease alcalase was used in water–organic cosolvents systems. The synthesis reaction conditions were optimized by examining the effects of several factors including water content, temperature, pH and reaction time on the yield of the synthesis product Bz-Arg-Gly-Asp(NH₂)OH. The optimum conditions are pH 8.0, 35 °C, in ethanol/Tris–HCl buffer system (85:15, v/v), 8 h with the tripeptide yield of 73.6%.     

Enhanced sensitivity and precision in an enzyme-linked immunosorbent assay with fluorogenic substrates compared with commonly used chromogenic substrates

Quantitative enzyme-linked immunosorbent assay (ELISA) is a widely used tool for analyzing biopharmaceutical and vaccine products. The superior sensitivity of the ELISA format is conferred by signal amplification through the enzymatic oxidation or hydrolysis of substrates to products with enhanced color or fluorescence. The extinction coefficient for a colored product or the quantum yield of a fluorescent product, coupled with the efficiency of the immobilized enzyme, is the determining factor for the sensitivity and precision of a given ELISA. The enhancement of precision and sensitivity using fluorogenic substrates was demonstrated in a direct-binding ELISA in a low-analyte concentration range compared with commonly used chromogenic substrates. The enhancement in precision was demonstrated quantitatively with lower coefficients of variation in measurements of signal intensities, approximately a five- to six-fold enhancement in signal-to-noise ratio at a given analyte concentration with fluorogenic substrates. Similarly, the amplitude of the enhancement in sensitivity, as reflected by relative limits of detection or quantitation, is approximately two- to five-fold when compared with commonly used chromogenic substrates. Additional advantages of a fluorescence-based ELISA format include the continuous monitoring of initial rates of enzymatic reactions, the measurement of fluorescence changes in the presence of particulate materials, the absence of a quench step, and a larger quantifiable analyte range.     

Development and validation of a fluorogenic assay to measure separase enzyme activity

Separase, an endopeptidase, plays a pivotal role in the separation of sister chromatids at anaphase by cleaving its substrate cohesin Rad21. Recent study suggests that separase is an oncogene. Overexpression of separase induces aneuploidy and mammary tumorigenesis in mice. Separase is also overexpressed and mislocalized in a wide range of human cancers, including breast, prostate, and osteosarcoma. Currently, there is no quantitative assay to measure separase enzymatic activity. To quantify separase enzymatic activity, we have designed a fluorogenic assay in which 7-amido-4-methyl coumaric acid (AMC)-conjugated Rad21 mitotic cleavage site peptide (Ac-Asp-Arg-Glu-Ile-Nle-Arg-MCA) is used as the substrate of separase. We used this assay to quantify separase activity during cell cycle progression and in a panel of human tumor cell lines as well as leukemia patient samples.     

Synthesis and evaluation of peptidic irreversible inhibitors of tissue transglutaminase

Herein we report the synthesis and the evaluation of eight novel compounds as irreversible inhibitors of transglutaminase (TGase). These compounds are based on a minimal peptidic scaffold shown previously [Chem. Biol. 2005, 12, 469–475] to confer affinity for the TGase active site and bear electrophilic groups such as ,β-unsaturated amide, chloroacetamide or maleimide; their general structure being Cbz-Phe-spacer-electrophile. The affinity conferred by the Cbz-Phe scaffold was determined by comparison to N-propylacrylamide and the length of the spacer was also varied to evaluate its importance. The inhibitory efficiencies (k_inact/K_I) of these compounds vary up to 10⁵ M⁻¹ min⁻¹, among the highest reported for derivatives based on this simple Cbz-Phe peptidic scaffold.     

Design, synthesis, and characterization of chromogenic substrates of coagulation factor XIIIa

A series of Glu(pNA)-containing peptides was designed to determine the activity of the transglutaminase factor XIIIa at 405 nm due to p-nitroaniline release. The most suitable substrate properties were found for peptides containing the Glu(pNA) residue in the second position from the N terminus. For the best substrate 12 (H-Tyr-Glu(pNA)-Val-Lys-Val-Ile-Gly-NH(2)), a k(cat)/K(m) value of 3531 s(-1)M(-1) was found. Although the k(cat)/K(m) values of the Glu(pNA) peptides are more than 100-fold reduced compared with the previously reported cleavage of natural glutamine-containing substrates such as α(2)-antiplasmin and β-casein, these chromogenic substrates can be useful tools for convenient determination of FXIII-A(2)* activity e.g., for in vitro inhibitor screening. As an example, peptide 12 was used to characterize the inhibition of FXIII-A(2)* by the well-known irreversible inhibitor iodoacetic acid.     

Synthesis and conformational characterization of the epidermal growth factor-like domain of blood coagulation factor IX carrying xylosyl-glucose

Solid-phase synthesis of glycopeptide generally requires the protection of both peptide side chains and hydroxyl groups of the carbohydrate portion. However, if the mild coupling conditions are used, the protection of the carbohydrate portion can be omitted. In this paper, we demonstrated it by the synthesis of Fmoc-serine carrying unmasked xylosyl glucose followed by the solid-phase synthesis of epidermal growth factor (EGF)-like domain of factor IX (45-87) using the unit. The product was well characterized by enzymatic digestion, amino acid analysis and mass spectrometry. The secondary structure of the product as well as glucosylated and non-glycosylated EGF-like domain was characterized by circular dichroism (CD) spectroscopy.     

Internally quenched fluorogenic protease substrates: Solid-phase synthesis and fluorescence spectroscopy of peptides containing ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs

Summary A general procedure, using the commonly employed solid-phase peptide synthesis methodology for obtaining internally quenched fluorogenic peptides with ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs, is presented. The essential feature of this procedure is the synthesis of an N -Boc or-Fmoc derivative of glutamic acid with the -carboxyl group bound to N-(2,4-dinitrophenyl)-ethylenediamine (EDDnp), which provides the quencher moiety attached to the C-terminus of the substrate. The fluorescent donor group, ortho-aminobenzoic acid (Abz), is incorporated into the resin-bound peptide in the last coupling cycle. Depending on the resin type used, Abz-peptidyl-Gln-EDDnp or Abz-peptidyl-Glu-EDDnp is obtained. Using the procedure described above, substrates for human renin and tissue kallikreins were synthesised. Spectrofluorimetric measurements of Abz bound to the -amino group of proline showed that strong quenching of Abz fluorescence occurs in the absence of any acceptor group.     

Amino acid and peptide derivatives of dimethyl 5-aminoisophthalate as fluorogenic substrates for proteinases

A number of amino acid and peptide derivatives of the fluorophore, dimethyl 5-aminoisophthalate have been synthesized, characterized and tested as substrates for the plant cysteine proteinases papain, ficin and bromelain. In every case, replacement of alanine by citrulline, in the position adjacent to the dimethyl 5-aminoisophthalate resulted in a higher rate of hydrolysis. The partly deprotected dipeptide derivative dimethyl phenylalanylcitrulline-5-aminoisophthalate was hydrolysed most rapidly of all the compounds tested, and on this basis may provide a useful substrate for the detection and quantitative assay of these enzymes.     

Characterization of monoacylglycerol acyltransferase 2 inhibitors by a novel probe in binding assays

Monoacylglycerol acyltransferase 2 (MGAT2) is a membrane-bound lipid acyltransferase that catalyzes the formation of diacylglycerol using monoacylglycerol and fatty acyl CoA as substrates. MGAT2 is important for intestinal lipid absorption and is an emerging target for the treatment of metabolic diseases. In the current study, we identified and characterized four classes of novel MGAT2 inhibitors. We established both steady state and kinetic binding assay protocols using a novel radioligand, [³H]compound A. Diverse chemotypes of MGAT2 inhibitors were found to compete binding of [³H]compound A to MGAT2, indicating the broad utility of [³H]compound A for testing various classes of MGAT2 inhibitors. In the dynamic binding assays, the kinetic values of MGAT2 inhibitors such as K_on, K_off, and T_1/2 were systematically defined. Of particular value, the residence times of inhibitors on MGAT2 enzyme were derived. We believe that the identification of novel classes of MGAT2 inhibitors and the detailed kinetic characterization provide valuable information for the identification of superior candidates for in vivo animal and clinical studies. The current work using a chemical probe to define inhibitory kinetics can be broadly applied to other membrane-bound acyltransferases.     

Productive recognition of factor IX by factor XIa exosites requires disulfide linkage between heavy and light chains of factor XIa

In the intrinsic pathway of blood coagulation factor XIa (FXIa) activates factor IX (FIX) by cleaving the zymogen at Arg(145)-Ala(146) and Arg(180)-Val(181) bonds releasing an 11-kDa activation peptide. FXIa and its isolated light chain (FXIa-LC) cleave S-2366 at comparable rates, but FXIa-LC is a very poor activator of FIX, possibly because FIX undergoes allosteric modification on binding to an exosite on the heavy chain of FXIa (FXIa-HC) required for optimal cleavage rates of the two scissile bonds of FIX. However preincubation of FIX with a saturating concentration of isolated FXIa-HC did not result in any potentiation in the rate of FIX cleavage by FXIa-LC. Furthermore, if FIX binding via the heavy chain exosite of FXIa determines the affinity of the enzyme-substrate interaction, then the isolated FXIa-HC should inhibit the rate of FIX activation by depleting the substrate. However, whereas FXIa/S557A inhibited FIX activation of by FXIa, FXIa-HC did not. Therefore, we examined FIX binding to FXIa/S557A, FXIa-HC, FXIa-LC, FXIa/C362S/C482S, and FXIa/S557A/C362S/C482S. The heavy and light chains are disulfide-linked in FXIa/S557A but not in FXIa/C362S/C482S and FXIa/S557A/C362S/C482S. In an ELISA assay only FXI/S557A ligated FIX with high affinity. Partial reduction of FXIa/S557A to produce heavy and light chains resulted in decreased FIX binding, and this function was regained upon reformation of the disulfide linkage between the heavy and the light chains. We therefore conclude that substrate recognition by the FXIa exosite(s) requires disulfide-linked heavy and light chains.     

Establishment of a novel high-throughput screening method for the detection and quantification of L-phosphinothricin produced by a biosynthesis approach

A novel and simple methodology for the detection of phosphinothricin produced by a biosynthesis approach in 96-well microtiter plates was developed based on the fluorometric determination of an isoindole derivative. The assay method to determine the generation of L-phosphinothricin was conducted with the help of a derivatization reaction. The linear detection range of the method was demonstrated to be from 0.74 to 100 μg ml⁻¹ for the catalysis by resting cells and from 1.6 to 100 μg ml^-1 for the catalysis by secretory enzymes. Meanwhile, the value of the relative standard deviation was less than 2.2% and the recovery was 99–101% of true value. Based on the method constructed in this study, one bacterial strain, Kluyvera cryocrescens ZJB-17005, with high stereoselectivity (>99%) and excellent yield (45%) was isolated from 13,284 strains in the production of L-phosphinothricin by a biosynthesis approach.     

Synthesis,characterization and biocompatibility of PEGA resins

Three types of beaded polyethylene glycol polyacrylamide copolymers (PEGA) with a high content of polyethylene glycol (PEG) were synthesized by inverse suspension polymerization and characterized for peptide synthesis and with respect to their physical properties. Several peptides of high purity have been synthesized on the resin. The properties which were determined were loading of amino group, swelling, bead size distribution, porosity, flexibility and compatibility with active biomolecules. A loading of 0.35 mmol/g has been obtained and the swelling was excellent in solvents of various polarities ranging from water to dichloromethane. The ¹³C-NMR T₁-relaxation times of a resin containing a peptide were determined in DMSO-d₆ and the resin was found to exhibit a behaviour similar to the components in free solution.     

S1 pocket fingerprints of human and bacterial methionine aminopeptidases determined using fluorogenic libraries of substrates and phosphorus based inhibitors

Methionyl aminopeptidases (MetAPs) are metallo-dependent proteases responsible for removing of N-terminal methionine residue of peptides and proteins during protein maturation and activation. In this report we use a comprehensive strategy to screen the substrate specificity of three methionyl aminopeptidases: Homo sapiens MetAP-1, Homo sapiens MetAP-2 and Escherichia coli MetAP-1. By utilizing a 65-membered fluorogenic substrate library consisting of natural and unnatural amino acids we established detailed substrate preferences of each enzyme in the S1 pocket. Our results show that this pocket is highly conserved for all investigated MetAPs, very stringent for methionine, and that several unnatural amino acids with methionine-like characteristics were also well hydrolyzed by MetAPs. The substrate-derived results were verified using several phosphonate and phosphinate-based inhibitors.