Cell in situ zymography: an in vitro cytotechnology for localization of enzyme activity in cell culture

In situ zymography is a unique technique for detection and localization of enzyme?Csubstrate interactions majorly in histological sections. Substrate with quenched fluorogenic molecule is incorporated in gel over which tissue sections are mounted and then incubated in buffer. The enzymatic activity is observed in the form of fluorescent signal. With the advancements in the field of biological research, use of in vitro cell culture has become very popular and holds great significance in multiple fields including inflammation, cancer, stem cell biology and the still emerging 3-D cell cultures. The information on analysis of enzymatic activity in cell lines is inadequate presently. We propose a single-step methodology that is simple, sensitive, cost-effective, and functional to perform and study the ??in position?? activity of enzyme on substrate for in vitro cell cultures. Quantification of enzymatic activity to carry out comparative studies on cells has also been illustrated. This technique can be applied to a variety of enzyme classes including proteases, amylases, xylanases, and cellulases in cell cultures.     

Metabolic mapping of proteinase activity with emphasis on in situ zymography of gelatinases: review and protocols.

Proteases are essential for protein catabolism, regulation of a wide range of biological processes, and in the pathogenesis of many diseases. Several techniques are available to localize activity of proteases in tissue sections or cell preparations. For localization of the activity of matrix metalloproteinases, in situ zymography was introduced some decades ago. The procedure is based on zymography using SDS polyacrylamide gels containing gelatin, casein, or fibrin as substrate. For in situ zymography, either a photographic emulsion containing gelatin or a fluorescence-labeled proteinaceous macromolecular substrate is brought into contact with a tissue section or cell preparation. After incubation, enzymatic activity is revealed as white spots in a dark background or as black spots in a fluorescent background. However, this approach does not allow precise localization of proteinase activity because of limited sensitivity. A major improvement in sensitivity was achieved with the introduction of dye-quenched (DQ-)gelatin, which is gelatin that is heavily labeled with FITC molecules so that its fluorescence is quenched. After cleavage of DQ-gelatin by gelatinolytic activity, fluorescent peptides are produced that are visible against a weakly fluorescent background. The incubation with DQ-gelatin can be combined with simultaneous immunohistochemical detection of a protein on the same section. To draw valid conclusions from the findings with in situ zymography, specific inhibitors need to be used and the technique has to be combined with immunohistochemistry and zymography. In that case, in situ zymography provides data that extend our understanding of the role of specific proteinases in various physiological and pathological conditions.     

Development of inductively coupled plasma-mass spectrometry-based protease assays

Rapid, sensitive, and quantitative assays for proteases are important for drug development and in the diagnosis of disease. Here an assay for protease activity that uses inductively coupled plasma-mass spectrometry (ICP-MS) detection is described. Peptidic α-chymotrypsin substrates were synthesized containing a lanthanide ion chelate at the N terminus to provide a distinct elemental tag. A biotin label was appended to the C terminus of the peptide, allowing separation of uncleaved peptide from the enzymatic digestion. The enzyme activity was determined by quantifying the lanthanide ion signal of the peptide cleavage products by ICP-MS. Biotinylated substrates synthesized include Lu-DTPA-Asp-Leu-Leu-Val-Tyr∼Asp-Lys(biotin) and Lu-DTPA-βAla-βAla-βAla-βAla-Gly-Ser-Ala-Tyr∼Gly-Lys-Arg-Lys(biotin)-amide. Parallel assays with a commercially available fluorogenic substrate (Suc-AAPF-AMC) for α-chymotrypsin were performed for comparison. Using the ICP-MS assay, enzyme concentrations as low as 2 pM could be readily detected, superior to the detection limit of an assay using the α-chymotrypsin fluorogenic substrate (Suc-AAPF-AMC). Furthermore, we demonstrated the use of this approach to detect chymotrypsin activity in HeLa cell lysates.     

Double-layer fluorescent zymography for processing protease detection

We have developed a novel double-layer zymographic method for the detection of specific processing proteases of a target proprotease using a specific fluorescent substrate. The target processing proteases were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the gel was subsequently incubated with the target proenzyme used as the substrate. A cellulose acetate membrane was immersed in 10% glycerol and then soaked in the fluorescent substrate solution. The slab gel of the processing protease was covered with the fluorescent substrate membrane, making a double layer. The double layer was incubated at 37 degrees C, and the released fluorescent band, in which the processing protease was located, was detected using UV light. The advantages of the double-layer fluorescent zymographic method are as follows: (i) the specific detection of target proprotease using a specific substrate, (ii) a relatively rapid and sensitive method, (iii) effective detection using small amounts of crude material, and (iv) wide applications that include the detection of processing proteases and activators for target proteases. Typical examples used for the detection of the processing proteases, such as plasminogen activator, chymotrypsinogen activator, procaspase-3 processing protease and caspase-3 activators, using this new method are described in this article.     

One- and two-dimensional SDS-PAGE zymography with quenched fluorogenic substrates provides identification of biological fluid proteases by direct mass spectrometry

We describe a simple and direct zymographic method for the detection of proteases using quenched fluorescent substrates. The proteases were separated using one- and two-dimensional electrophoresis, and the gel subsequently was incubated with the quenched fluorescent substrate. After a short incubation, the released fluorescence allowed the localization of the proteases directly using UV light. The protease spots could then be cut directly from the gel and processed for identification by mass spectrometry. This method could easily be used to develop or test whether a substrate is specific or not and also to detect the proteases that are able to cleave this substrate in a complex biological fluid. This also allowed direct identification of proteases without complex purification.     

Peptide substrate for caspase-3 with 2-aminoacridone as reporting group

Synthesis and properties of a new fluorescent/fluorogenic substrate Ac-DEVD-AMAC for caspase-3 are reported. The substrate is obtained by conventional Fmoc-based solid phase peptide synthesis and its properties are investigated with regard to fluorescence, sensitivity, applicability and kinetic constants. A non-traditional approach to assay the proteases activity using 2-aminoacridone labeled peptides is proposed. This approach utilizes the decrease of fluorescence intensity of a sample as a measure for the enzyme activity.     

A bioluminescent assay for the sensitive detection of proteases

A bioluminescent general protease assay was developed using a combination of five luminogenic peptide substrates. The peptide-conjugated luciferin substrates were combined with luciferase to form a homogeneous, coupled-enzyme assay. This single-reagent format minimized backgrounds, gave stable signals, and reached peak sensitivity within 30 min. The bioluminescent assay was used to detect multiple proteases representing serine, cysteine, and metalloproteinase classes. The range of proteases detected was broader and the sensitivity greater, when compared with a standard fluorescent assay based on cleavage of the whole protein substrate casein. Fifteen of twenty proteases tested had signal-to-background ratios >10 with the bioluminescent method, compared with only seven proteases with the fluorescent approach. The bioluminescent assay also achieved lower detection limits (≤100 pg) than fluorescent methods. During protein purification processes, especially for therapeutic proteins, even trace levels of contamination can impact the protein's stability and activity. This sensitive, bioluminescent, protease assay should be useful for applications in which contaminating proteases are detrimental and protein purity is essential.     

Evaluation of the CFP-substrate-YFP system for protease studies: advantages and limitations

A protease can be defined as an enzyme capable of hydrolyzing peptide bonds. Thus, characterization of a protease involves identification of target peptide sequences, measurement of activities toward these sequences, and determination of kinetic parameters. Biological protease substrates based on fluorescent protein pairs, which allow for use of fluorescence resonance energy transfer (FRET), have been recently developed for in vivo protease activity detection and represent a very interesting alternative to chemical substrates for in vitro protease characterization. Here, we analyze a FRET system consisting of cyan and yellow fluorescent proteins (CFP and YFP, respectively), which are fused by a peptide linker serving as protease substrate. Conditions for CFP-YFP fusion protein production in Escherichia coli and purification of proteins were optimized. FRET between CFP and YFP was found to be optimum at a pH between 5.5 and 10.0, at low concentrations of salt and a temperature superior to 25 degrees C. For efficient FRET to occur, the peptide linker between CFP and YFP can measure up to 25 amino acids. The CFP-substrate-YFP system demonstrated a high degree of resistance to nonspecific proteolysis, making it suitable for enzyme kinetic analysis. As with chemical substrates, substrate specificity of CFP-substrate-YFP proteins was tested towards different proteases and kcat/Km values were calculated.     

Detection of proteolytic activity by fluorescent zymogram in-gel assays

Proteases are involved in the regulation of many biological functions. This study describes a novel method for detecting protease activity by fluorescent zymogram in-gel protease assays, using SDS polyacrylamide gels copolymerized with a peptide-MCA (4-methyl-coumaryl-7-amide) substrate. This method allows simultaneous determination of protease cleavage specificity and molecular weight. Trypsin was electrophoresed in SDS polyacrylamide gels copolymerized with Boc-Gln-Ala-Arg-MCA, the gel was then incubated in assay buffer, and trypsin cleavage of the peptide-MCA substrate generated fluorescent AMC (7-amino-4-methyl-coumarin), which was subsequently detected under UV transillumination. Chymotrypsin activity was detected in gels copolymerized with Suc-Ala-Ala-Pro-Phe-MCA substrate. Selective detection of these proteases was demonstrated by the absence of trypsin activity in gels containing the chymotrypsin substrate, and the lack of chymotrypsin activity in gels containing the trypsin substrate. Detection of proteolytic activity from secretory vesicles of adrenal medulla (chromaffin granules) was observed with the trypsin substrate, Z-Phe-Arg-MCA, but not with the chymotrypsin substrate. Overall, this sensitive fluorescent zymogram in-gel protease assay method can be used for rapid determination of protease cleavage specificity and enzyme molecular weight in biological samples. This assay should be useful for many research disciplines investigating the role of the many proteases that control cellular functions.     

Improvements of TArgeted multiplex mass spectrometry IMaging

MALDI mass spectrometers have become popular tools for imaging histological sections. Currently this technology is primarily used for imaging naturally occurring molecules. Here we report on the improvement of TArgeted multiplex MS IMaging (TAMSIM) technology. For TAMSIM we attach photocleavable mass tags to antibodies. Staining histological sections is done analogously to standard immunohistochemical procedures with chemiluminescent or fluorescent detection with the sole difference that multiple antibodies each with a distinct mass tag are used in a single reaction. Mass tags are released from their respective antibodies by a laser pulse at 355 nm without added matrix. After scanning, MS images are created for each tag mass. The enhancements of TAMSIM presented here relate to four elements, the use of an improved generation of tags, their conjugation directly to primary antibodies, the comparison of fresh frozen sections with paraffin embedded ones for the TAMSIM imaging technology and finally, the increase of multiplex detection. Sections of healthy human pancreatic tissue were imaged to visualize different specific biomarkers (synaptophysin, chromogranin, insulin, calcitonin, somatostatin) in neuroendocrine cells of Langerhans islets. The aim was to localize these biomarkers on the tissue sections simultaneously.     

Homogeneous real-time detection and quantification of nucleic acid amplification using restriction enzyme digestion

A method for real-time fluorescent detection and quantification of nucleic acid amplification using a restriction endonuclease was developed. In this homogeneous system detection is mediated by a primer containing a reporter and quencher moiety at its 5' terminus separated by a short section of DNA encoding a restriction enzyme recognition sequence. In the single stranded form, the signal from the fluorescent reporter is quenched due to fluorescence resonance energy transfer. However, as the primer becomes incorporated into a double stranded amplicon, a restriction enzyme present in the reaction cleaves the DNA linking the reporter and quencher, allowing unrestricted fluorescence of the reporter. To test this system, a primer specific for the E6 gene of human papilloma virus (HPV) 16 was combined with the cleavable energy transfer label and used to amplify HPV16 positive DNA. In the presence of the thermally stable restriction enzyme BstNI, the reporter system was found to generate a fluorescent signal in proportion to the amount of template DNA. In addition to this direct format, the reporter primer was also used to monitor and quantify the amplification of other sequences. This was accomplished by using primers that contain a tag sequence complementary to the reporter oligonucleotide.     

Visual detection of peptidase activity using fluorogenic substrates in a microtiter plate assay

A simple, inexpensive, and sensitive assay for peptidase activity has been devised. The assay was performed in a microtiter plate and was based on fluorogenic peptide substrates, many of which are commercially available. 7-Amino-4-methyl coumarin the fluorescent product liberated during an incubation period of between 1 and 16 h, was detected by inspection of the plate under ultraviolet light of wavelength 356 nm. A fluorometer was not required. Using alpha-chymotrypsin as a model enzyme, with succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4-methyl-coumaryl-7-amide as substrate, it was shown that as little as 4 fmol of enzyme could be detected. The method was non-quantitative and was particularly suited to location of enzyme activity in fractions during a purification procedure. The validity of the assay was demonstrated by detection of activity of a known enzyme, alpha-chymotrypsin, after its purification by size-exclusion high-performance liquid chromatography. The method was used to locate two forms of aminopeptidase activity, in fractions from size-exclusion chromatography of an extract from reproductive tissue of Helix aspersa, using L-leucine 4-methyl-coumaryl-7-amide as substrate.     

Efficient near infrared fluorescence detection of elastase enzyme using peptide-bound unsymmetrical squaraine dye

Extended wavelength analyte-responsive fluorescent probes are highly desired for the imaging applications owing to their deep tissue penetration, and minimum interference from autofluorescence by biomolecules. Near infra-red (NIR) sensitive and self-quenching fluorescent probe based on the dye-peptide conjugate (SQ 1 PC) was designed and synthesized by facile and efficient one-pot synthetic route for the detection of Elastase activity. In the phosphate buffer solution, there was an efficient quenching of fluorescence of SQ 1 PC (86%) assisted by pronounced dye-dye interaction due to H-aggregate formation. Efficient and fast recovery of this quenched fluorescence of SQ 1 PC (> 50% in 30 s) was observed on hydrolysis of this peptide-dye conjugate by elastase enzyme. Presently designed NIR sensitive self-quenching substrate offers the potential application for the detection of diseases related to proteases by efficient and fast detection of their activities.     

Substrate modulation of enzyme activity in the herpesvirus protease family

The herpesvirus proteases are an example in which allosteric regulation of an enzyme activity is achieved through the formation of quaternary structure. Here, we report a 1.7 A resolution structure of Kaposi's sarcoma-associated herpesvirus protease in complex with a hexapeptide transition state analogue that stabilizes the dimeric state of the enzyme. Extended substrate binding sites are induced upon peptide binding. In particular, 104 A2 of surface are buried in the newly formed S4 pocket when tyrosine binds at this site. The peptide inhibitor also induces a rearrangement of residues that stabilizes the oxyanion hole and the dimer interface. Concomitant with the structural changes, an increase in catalytic efficiency of the enzyme results upon extended substrate binding. A nearly 20-fold increase in kcat/KM results upon extending the peptide substrate from a tetrapeptide to a hexapeptide exclusively due to a KM effect. This suggests that the mechanism by which herpesvirus proteases achieve their high specificity is by using extended substrates to modulate both the structure and activity of the enzyme.     

A dual-purpose synthetic colloidal platform for protease mapping: substrate profiling for Dengue and West Nile virus proteases

In a proof of concept study, we created a small focused fluorescent hexapeptide library onto 14 multiplexed barcoded sets of silica particles to probe the substrate recognition specificity of West Nile and Dengue virus proteases. A flow cytometric analysis demonstrated that the optical signature of each bead population remained distinguishable throughout the solid-phase peptide synthesis and proteolytic assay. As expected, both proteases displayed a narrow specificity for lysine and arginine residues in the P₁ and P₂ substrate positions. This open-ended platform enables the fast and simultaneous identification of peptide substrates and is applicable to other proteases.     

Enzyme histochemical discrimination between tryptase and chymase in mast cells of human gut

We tested four synthetic substances for their histochemical value to demonstrate the catalytic activities of chymase or tryptase in mast cells in sections of human gut. Both Suc-Ala-Ala-Phe-4 methoxy-2-naphthylamide (MNA) and N-acetyl-L-methionine-alpha-naphthyl ester (alpha-N-O-Met) reacted with chymase but not tryptase in mast cells. Conversely, D-Val-Leu-Arg-MNA and Z-Ala-Ala-Lys-MNA were hydrolyzed by mast cell tryptase but not chymase. These results were confirmed by use of two inhibitors of chymotrypsin-like activity, chymostatin and Z-Gly-Leu-Phe-chloromethyl ketone (CK) and two inhibitors of trypsin-like activity, Tos-Lys-CK and D-Val-Leu-Arg-CK. Excellent staining reactions were obtained on cryostat sections of unfixed or aldehyde-fixed tissues and on paraffin sections of Carnoy-fixed tissues. For chymase, however, Suc-Ala-Ala-Phe-MNA is preferred on cryostat sections because it is more specific. On paraffin sections alpha-N-O-Met is preferred because other cells are not then stained. For tryptase, Z-Ala-Ala-Lys-MNA was more selective and more specific and is the preferred general purpose substrate on cryostat sections of aldehyde-fixed tissues and for paraffin sections. D-Val-Leu-Arg-MNA is the preferred substrate for cryostat sections of unfixed tissue. Only a limited number of mast cells showed a reaction for chymase, and these occurred mainly in the submucosa. All mast cells, however, gave a reaction for tryptase, and we recommend the use of either substrate for this enzyme for routine detection of mast cells in human tissues. Double staining for the two main mast cell proteases is most conveniently undertaken on paraffin sections of Carnoy-fixed tissues using MNA substrates for tryptase and alpha-N-O-Met for chymase.     

Identification of preferred substrate sequences for transglutaminase 1--development of a novel peptide that can efficiently detect cross-linking enzyme activity in the skin

Transglutaminase 1 (TGase 1) is an essential enzyme for cornified envelope formation in stratified squamous epithelia. This enzyme catalyzes the cross-linking of glutamine and lysine residues in structural proteins in differentiating keratinocytes. To gain insight into the preferred substrate structure of TGase 1, we used a phage-displayed random peptide library to screen primary amino acid sequences that are preferentially selected by human TGase 1. The peptides selected as glutamine donor substrate exhibited a marked tendency in primary structure, conforming to the sequence: QxK/RpsixxxWP (where x and psi represent non-conserved and hydrophobic amino acids, respectively). Using glutathione S-transferase (GST) fusion proteins of the selected peptides, we identified several sequences as preferred substrates and confirmed that they were isozyme-specific. We generated GST-fused alanine mutants of the most reactive sequence (K5) to determine the residues that were critical for reactivity. Even in peptide form, K5 appeared to have high and specific reactivity as substrate. In situ analysis of mouse skin sections using fluorescence-conjugated K5 peptide resulted in detection of TGase 1 activity with high sensitivity, but no signal was detected in a TGase 1-null mouse. In conclusion, we were successful in generating a novel substrate peptide for sensitive detection of endogenous TGase 1 activity in the skin.     

Evaluation of different chemical strategies for conjugation of oligonucleotides to peptides

We developed novel assays for high-throughput detection of one or many kinases or proteases. The assays use hundreds of different peptide substrates, each covalently linked to an oligonucleotide tag. After incubation with sample, the pool of substrates is hybridized to a microarray containing oligonucleotides complementary to the tag sequences. We screened several specific chemistries for the conjugation based on the following criteria: easy derivatization of oligonucleotides and peptides; high efficiency of the conjugation reaction; good stability of the conjugates; and satisfactory conjugate performance in our assays. We have validated selected method during the successful generation of thousands oligonucleotide-peptide conjugates.     

Deciphering Enzyme Function Using Peptide Arrays

Enzymes are key molecules in signal-transduction pathways. However, only a small fraction of more than 500 human kinases, 300 human proteases and 200 human phosphatases is characterised so far. Peptide microarray based technologies for extremely efficient profiling of enzyme substrate specificity emerged in the last years. This technology reduces set-up time for HTS assays and allows the identification of downstream targets. Moreover, peptide microarrays enable optimisation of enzyme substrates. Focus of this review is on assay principles for measuring activities of kinases, phosphatases or proteases and on substrate identification/optimisation for kinases. Additionally, several examples for reliable identification of substrates for lysine methyl-transferases, histone deacetylases and SUMO-transferases are given. Finally, use of high-density peptide microarrays for the simultaneous profiling of kinase activities in complex biological samples like cell lysates or lysates of complete organisms is described. All published examples of peptide arrays used for enzyme profiling are summarised comprehensively.     

Improved detection of botulinum neurotoxin type A in stool by mass spectrometry

Botulinum neurotoxins (BoNTs) are the most toxic substances known to humankind. Rapid and sensitive detection of BoNTs is necessary for timely clinical confirmation of the disease state in botulism. BoNTs cleave proteins and peptide mimics at specific sites. A mass spectrometry (MS)-based method, Endopep–MS, can detect these cleavages and has detection limits of 0.05–0.5 mouse LD₅₀ (U) in serum, depending on the BoNT serotypes. In this method, the products generated from cleavage of peptide substrates using antibody affinity-purified toxins are detected by MS. Nonspecific bound endogenous proteases or peptidases in stool can coextract with the toxin, cleaving the peptide substrates and reducing the sensitivity of the method. Here we report a method to reduce nonspecific substrate cleavage by reducing stool protease coextraction in the Endopep–MS assay.