ARTICLE |
Correspondence to: Cornelis J.F. Van Noorden, Dept. of Cell Biology and Histology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. E-mail: c.j.vannoorden@amc.uva.nl
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Summary |
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Fluorogenic substrates [Ala-Pro]2-cresyl violet and Ala-Pro-rhodamine 110 have been tested for microscopic detection of protease activity of dipeptidyl peptidase IV (DPPIV) in living cells. DPPIV activity is one of the many functions of the multifunctional or moonlighting protein CD26/DPPIV. As a model we used Jurkat cells, which are T-cells that lack CD26/DPPIV expression, and CD26/DPPIV-transfected Jurkat cells. Ala-Pro-rhodamine 110 is not fluorescent, but after proteolytic cleavage rhodamine 110 fluoresces. [Ala-Pro]2-cresyl violet is fluorescent by itself but proteolytic cleavage into cresyl violet induces a shift to longer wavelengths. This phenomenon enables the simultaneous determination of local (intracellular) substrate and product concentrations, which is important for analysis of kinetics of the cleavage reaction. [Ala-Pro]2-cresyl violet, but not Ala-Pro-rhodamine 110, appeared to be specific for DPPIV. When microscopic analysis is performed on living cells during the first minutes of the enzyme reaction, DPPIV activity can be precisely localized in cells with the use of [Ala-Pro]2-cresyl violet. Fluorescent product is rapidly internalized into submembrane granules in transfected Jurkat cells and is redistributed intracellularly via internalization pathways that have been described for CD26/DPPIV. We conclude that [Ala-Pro]2-cresyl violet is a good fluorogenic substrate to localize DPPIV activity in living cells when the correct wavelengths are used for excitation and emission and images are captured in the early stages of the enzyme reaction. (J Histochem Cytochem 51:959968, 2003)
Key Words: living cell cytochemistry, protease, metabolic mapping, biocomplexity, CD26/DPPIV, Jurkat cells, confocal scanning laser, microscopy, fluorescence microscopy
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Introduction |
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NOW that genomes of species are becoming elucidated and proteomic analyses in health and disease are under way, analysis of functions of proteins is becoming more important than ever (
Various types of synthetic fluorogenic substrates are available for detection of activity of proteases in living cells (for review see
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Proline is a unique amino acid because of its cyclic structure. This specific conformation of proline imposes many restrictions on cleavage of peptides and proteins that contain proline. A large series of physiologically important biomolecules contain proline in the penultimate position and their biological properties are highly regulated by this proline motif. Only a limited number of proteases can cleave proline-containing peptides (
In this study we compared the reactivity of two synthetic substrates that contain the same amino acid sequence but different fluorogenic leaving groups, cresyl violet (
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Materials and Methods |
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Jurkat Cell Lines
Jurkat cells (clone E6-1; American Type Culture Collection, Manassas, VA), which lack CD26/DPPIV expression, were used as well as Jurkat cells transfected with CD26/DPPIV (
Western Blotting and Zymography of DPPIV Activity
To test specificity of substrates, samples containing soluble CD26/DPPIV (sCD26/DPPIV) derived from human seminal fluid were run on polyacrylamide gels and submitted to Western blotting or were incubated in the presence of a series of synthetic DPPIV substrates. sCD26/DPPIV was enriched by isolating prostasomes from human seminal fluid, as described by
DPPIV activity was detected with the following substrates: 20 µM [Ala-Pro]2-cresyl violet (Enzyme Systems Products and Prototek, Livermore, CA;
Samples were also subjected to Western blotting to determine CD26/DPPIV protein expression. Proteins were blotted at 4C and 100 V to nitrocellulose paper for 1 hr. After washing in PBS containing 0.05% Tween, blots were blocked overnight with 5% BSA in PBS. Blots were incubated with anti-CD26 antibody Ta1 (Central Laboratory for Blood Transfusion; Amsterdam, The Netherlands) at a dilution of 1:100 in the blocking buffer containing 0.05% Tween for 1 hr, then washed twice in PBS containing 0.05% Tween, followed by 1-hr incubation with monoclonal horseradish peroxidase-conjugated goat anti-mouse IgG (Nordic; Tilburg, The Netherlands) in a dilution of 1:200 in blocking buffer. Then blots were washed again in PBS. Finally, after 10 min of incubation with Lumi-Light Western blotting substrate (Roche Diagnostics; Mannheim, Germany), chemiluminescence was detected by the Lumi-Imager (Roche Diagnostics).
Fluorescence Spectra of [Ala-Pro]2-Cresyl Violet Substrate and Cresyl Violet Product
Fluorometric analysis of concentrations of [Ala-Pro]2-cresyl violet and cresyl violet acetate (Enzyme Systems Products) was performed on a LS 50 fluorescence spectrometer (PerkinElmer; Gouda, The Netherlands). [Ala-Pro]2-cresyl violet (20 µM) and cresyl violet acetate (20 µM) were measured separately and mixed in 1:1 and 1:4 ratios. Excitation was performed at 496 nm and 591 nm and fluorescence emission was monitored at wavelengths ranging from 500 nm to 700 nm.
Thin-Layer Chromatography of [Ala-Pro]2-Cresyl Violet Substrate and Cresyl Violet Product
To demonstrate fluorescent components in batches of substrate and product, separation by thin-layer chromatography (TLC) was performed, using silica gel-coated TLC plates (Merck; Darmstadt, Germany) as the stationary phase and methanol as the mobile phase. Equal amounts of substrate and product molecules were dissolved in methanol and applied to TLC plates. The plates were placed upright in running fluid and ran until the front of the running fluid had reached the end of the plate. The plates were dried and stored for further analysis. Images of the plates were made using white light and a digital camera (Coolpix; Nikon, Tokyo, Japan) to demonstrate the color change from orange to violet as is also observed with the naked eye when living cells are incubated with the substrate in a cuvette. Then the plates were illuminated with UVA light (320380 nm), and photographed with a UV-blocking filter (>500 nm) using the same camera.
Fluorospectrometric Analysis of DPPIV Activity
Living Jurkat cells and CD26/DPPIV-transfected Jurkat cells were harvested and DPPIV activity was determined by fluorospectrometry. Before DPPIV activity measurements, cells were washed twice in cold PBS. Intact cells were kept on ice before mixing with the enzyme incubation media. Parts of the cells were lysed by ultrasonic treatment three times for 5 sec. Incubations were started at t=0 by suspending Jurkat cells or transfected Jurkat cells in prewarmed PBS supplemented with 1.7 mM CaCl2 and 1 mM MgCl2 at 37C containing 040 µM of the DPPIV substrate [Ala-Pro]2-cresyl violet or Ala-Pro-rhodamine 110. Actual amounts of ester bonds that are cleaved are twice as high as that of the free fluorochrome in the case of [Ala-Pro]2-cresyl violet, because two amino acid sequences are spliced off per cresyl violet fluorochrome (Fig 1). Incubations were carried out at 37C by using prewarmed PBS to which the substrate was added just before the start of the incubation. The cell suspension was kept on ice before the incubation and added 30 sec after the start of recording. For each measurement, 4 x 106 living cells, or its equivalent in cell lysates, were incubated in a final volume of 1200 µl prewarmed PBS containing 040 µM [Ala-Pro]2-cresyl violet or Ala-Pro-rhodamine 110 substrate. Fluorometric analysis was performed on an LS 50 fluorescence spectrometer under continuous magnetic stirring to keep cells in suspension. Cuvettes with an excitation light path of 1 cm and an emission light path of 4 mm were used. Excitation for cresyl violet was performed at 591 nm with a slit width of 10 nm and emission was detected at 628 nm with a slit width of 10 nm (
Confocal Microscopic Analysis of DPPIV Activity in Living Cells
Confocal analysis was performed to localize DPPIV-like activity in living Jurkat and transfected Jurkat cells on a Leica SP2 AOBS confocal microscope (Leica Microsystems; Mannheim, Germany). In case of the use of [Ala-Pro]2-cresyl violet, fluorescence of both substrate and product was analyzed with settings of the AOBS for 488-nm excitation of the substrate and 594-nm excitation of the product. Fluorescence was measured at 515576 nm and 613734 nm for substrate and product, respectively. Each focal plane was scanned in a sequential manner in time, xyt, or in volume, xyz, for end-point images. In case of the use of Ala-Pro-rhodamine 110, excitation was performed at 496 nm and emission was measured at 550580 nm. Living Jurkat cells were incubated on the stage of a confocal microscope in Dulbecco's modified medium containing 0.1 mg/ml Geneticin G418 and 1 mM glutamine and 10% FCS. This medium allowed prolonged incubations for longer periods of time without any significant cell damage. The suspended cells were incubated in 1 ml incubation medium on the microscope stage on a -irradiated glass bottom in a poly-L-lysine-coated petri dish (MatTek; Ashland, MA). The nonadherent blood cells tend to stick after a certain period of time to the poly-L-lysine-coated glass bottom. After allowing the cells to adhere for a few minutes, cells were selected in the transmission image mode using white light and brought into focus, and transmission images were made to check whether cells were not moving any more. Then substrate was added by carefully dropping the substrate on top of the cells, preventing the cells to drift out of focus or the field of observation. Then the substrate was added as 10 µl of a 100 x concentrated stock solution to the medium. The petri dishes were mounted in the specimen clamp on the stage of the microscope with the use of a circular rubber self-constructed O-ring. This enables the administration of the substrate directly on top of the cells, while the cells can be imaged continuously. The first fluorescence images were captured at 30 sec after the substrate was added in a single focal plane at a rate of 1.8 sec per scan. In total, 200 images were made in 6 min. Sequential scanning was performed to minimize crosstalk.
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Results |
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Reactivity of the various Ala-Pro-containing synthetic substrates with CD26/DPPIV was demonstrated with the use of an enriched fraction of sCD26/DPPIV that was submitted to native gel electrophoresis and Western blotting. Fig 2 demonstrates a similarly stained banding pattern with two major bands of DPPIV activity obtained with all substrates tested. Western blotting after staining with the anti-CD26/DPPIV antibody shows a similar banding pattern as well (Fig 2). These findings indicate that all substrates identify DPPIV activity and demonstrate the different isoforms of DPPIV/CD26 as they occur in prostasomes. It should be noted that intensities cannot be compared directly due to the limited number of excitation wavelengths available on the scanner used and thus to the more or less optimal excitation of the fluorophores.
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To characterize the shift in fluorescence that occurs after proteolytic cleavage of [Ala-Pro]2-cresyl violet into cresyl violet and Ala-Pro dipeptides, emission and excitation spectra were determined for both [Ala-Pro]2-cresyl violet substrate and cresyl violet product separately (Fig 3A and Fig 3B), and in mixtures with different ratios (1:1, 1:4; Fig 3C). Solutions of 20 µM were made of both substrate and product, and emission spectra were determined. When light of 480, 500, 520, or 540 nm was used for excitation, [Ala-Pro]2-cresyl violet showed one emission peak at 570 nm (Fig 3A). When excitation light of 560 or 580 nm was used, emission was at a higher wavelength but insignificant. When light of 480, 500, 520, 540, 560, or 580 nm was used for excitation, the proteolytic product cresyl violet was fluorescent with one emission peak only at 628 nm (Fig 3B). Because of the broad fluorescence spectrum of the substrate [Ala-Pro]2-cresyl violet, fluorescence of the product could not be detected specifically when excitation light was used at a wavelength <570 nm because the substrate caused too high levels of background fluorescence (Fig 3C). In practice, substrate concentrations are much higher than product concentrations in cells when initial stages of enzyme reactions are measured. On the other hand, the 570-nm fluorescence of [Ala-Pro]2-cresyl violet substrate can be used to determine amounts of substrate present in both cells and/or incubation medium to enable the determination of kinetics of [Ala-Pro]2-cresyl violet cleavage on the basis of local substrate concentrations and production of cresyl violet. The spectra illustrate that for specific measurement of fluorescence of the cresyl violet product at 628 nm, excitation at 591 nm must be used to avoid interference by fluorescence of [Ala-Pro]2-cresyl violet (Fig 3C). In conclusion, when an excitation wavelength of 591 nm is used, fluorescence at 628 nm is specific for the product. The substrate concentration can be measured using excitation at 488 nm and emission at 570 nm.
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Ala-Pro-rhodamine 110 substrate is not fluorescent, whereas the DPPIV reaction product, rhodamine 110, has an excitation peak at 491 nm and an emission peak at 529 nm (
Comparison of [Ala-Pro]2-cresyl violet and cresyl violet bands after TLC shows the color shift in both absorbance and fluorescence when the substrate is proteolytically cleaved (Fig 3D). A similar color change is observed when CD26/DPPIV is incubated with the substrate. Specificity of [Ala-Pro]2-cresyl violet and Ala-Pro-rhodamine 110 as substrates for CD26/DPPIV was demonstrated in intact and permeabilized wild-type Jurkat cells and CD26/DPPIV-transfected Jurkat cells by incubating the cells for 4 min in the presence of both substrates (Fig 4). A small increase in fluorescence over time was observed when both synthetic substrates, but particularly [Ala-Pro]2-cresyl violet, were incubated in the aqueous medium in the absence of cells, indicating spontaneous disintegration of the substrates. The increase in fluorescence over time in wild-type Jurkat cells was similar to the spontaneous disintegration of [Ala-Pro]2-cresyl violet, indicating that other DPPIV-like proteases did not cleave the substrate. CD26/DPPIV-transfected Jurkat cells produced significantly higher amounts of fluorescence at 628 nm in the presence of [Ala-Pro]2-cresyl violet. Fig 4B shows a similar production of fluorescence at 628 nm in intact and permeabilized transfected Jurkat cells against 20 µM [Ala-Pro]2-cresyl violet, again indicating that intracellular DPPIV-like proteases do not cleave the substrate.
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Wild-type Jurkat cells lacking CD26/DPPIV process Ala-Pro-rhodamine 110 at a considerable rate, but the reaction velocity in CD26/DPPIV-transfected Jurkat cells against 20 µM Ala-Pro-rhodamine 110 was considerably higher than in Jurkat cells lacking the enzyme (Fig 4C). Permeabilization increases the reaction rate in both wild-type Jurkat cells and transfected Jurkat cells, indicating that intracellular DPPIV-like proteases cleave the rhodamine 110-containing substrate (Fig 4D). Please note that inner filter effects did not play a significant role because all plots are linear with time, showing that saturation does not occur due to inner filter effects. Km values of cleavage of [Ala-Pro]2-cresyl violet and Ala-Pro-rhodamine 110 by intact living and permeabilized wild-type Jurkat cells and CD26/DPPIV-transfected Jurkat cells were similar in the range of 310 µM (Table 1). Remarkably, Vmax values of Ala-Pro-rhodamine 110 cleavage were similar in wild-type Jurkat cells and transfected Jurkat cells, and permeabilization of the cells increased Vmax almost threefold (Table 1). Please note that Vmax values are given in arbitrary fluorescence values, so the values for rhodamine 110 can be directly compared but not rhodamine 110 values with cresyl violet values.
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Confocal microscopical analysis of cleavage of [Ala-Pro]2-cresyl violet and Ala-Pro-rhodamine 110 in living wild-type Jurkat cells and CD26/DPPIV-transfected Jurkat cells is shown in Fig 5 and Fig 6. Accumulation of cresyl violet in time was observed only in transfected Jurkat cells (Fig 5) and not in Jurkat cells lacking CD26/DPPIV (Fig 6) in small vesicles near the plasma membrane during the first few minutes of incubation. After longer incubation periods, the fluorochrome was transported to other organelles in transfected Jurkat cells (Fig 7), mimicking the pathways of internalization that have been described for CD26/DPPIV (
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When Ala-Pro-rhodamine 110 was used as substrate, fluorescence occurred in both transfected and wild-type living Jurkat cells. Although fluorescence appeared to be more diffuse, accumulation was also observed in small vesicle-like structures near the plasma membrane (Fig 7).
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Discussion |
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For visualization of specific enzyme reactions in living cells in general and activity of CD26/DPPIV in particular, it has to be established whether other enzymes interfere in the reaction and whether substrate is available at all sites of the enzyme (
Surprisingly, the two substrates behaved in rather different ways with respect to specificity. Ala-Pro-rhodamine 110 appeared to react with other DPPIV activity homologues, such as lysosomal DPPII, DPPVIII, and attracktin, whereas [Ala-Pro]2-cresyl violet reacted with DPPIV only. The most likely explanation is that the catalytic cleft of DPPIV only is suitable to give access to cresyl violet. The 3D configuration of the tunnel in the molecule of CD26/DPPIV and activity homologues that gives access to the active site is rather tight (
Excitation and emission spectra of cresyl violet and rhodamine 110 are not the same. Therefore, differences in absolute amounts of product formation on the basis of increases in fluorescence cannot be compared. Probably, rhodamine 110 is excited more intensely than cresyl violet, because light of 488 nm is more intense than light at 591 nm. The illumination source is not similarly intense throughout the spectrum, as light (lasers) does not produce similar amounts of photons at each wavelength. Furthermore, hydrolysis of monosubstituted and bisubstituted fluorochromes is not comparable either, especially when over 15% of the substrate is hydrolyzed during an assay (
With respect to cytotoxicity, it can be stated that low amounts of excitation light should be used to avoid damage to the cells. Preferentially, laser power lower than 10 mW/cm2 should be used (E. Manders, personal communication). Illumination by laser light is more toxic for cells containing fluorochromes than cells without fluorochromes. Because of the lower energy of light at longer wavelengths, [Ala-Pro]2-cresyl violet is also to be preferred over Ala-Pro-rhodamine 110 because cresyl violet has to be excited at 591 nm and rhodamine at 488 nm.
A disadvantage of the cresyl violet-based substrate over the rhodamine-based substrate is the rather high instability of the former in aqueous solutions. However, the great advantage of [Ala-Pro]2-cresyl violet is its specificity for DPPIV activity. Wild-type Jurkat cells do not produce more fluorescence than incubation medium only, whereas CD26/DPPIV-transfected Jurkat cells produce distinctly more fluorescence. The same incubations were also performed on permeabilized cells. After sonification, cells were incubated as described above. Permeabilization of wild-type Jurkat cells and of CD26/DPPIV-transfected Jurkat cells did not lead to an additional increase in fluorescence. Apparently, [Ala-Pro]2-cresyl violet does not react with other DPPIV activity homologues.
In intact cells, green fluorescence of the [Ala-Pro]2-cresyl violet substrate is converted into red fluorescence of the liberated cresyl violet. Strikingly, damaged or apoptotic cells showed hardly any enzymatic activity but did accumulate [Ala-Pro]2-cresyl violet rapidly, as if the enzyme was already inactivated.
Although [Ala-Pro]2-cresyl violet is specific for DPPIV, localization of the cleavage product is another matter. Accumulation of the fluorochrome at first in small submembranous granules and later in larger intracellular compartments may be due to lipophilicity, the charge of cresyl violet, or transport in intact CD26/DPPIV-expressing cells, because intracellular localization patterns are in agreement with the recycling pathway of the M6P receptor, which can bind CD26/DPPIV and molecules associated with it (
Ala-Pro-rhodamine 110 also shows some spontaneous hydrolysis when dissolved in incubation medium. When incubations were performed with intact living Jurkat cells lacking CD26/DPPIV, hydrolysis of Ala-Pro-rhodamine 110 was distinctly higher, demonstrating its lack of specificity. CD26/DPPIV-positive transfected Jurkat cells produced an additional increase of fluorescence over time. Permeabilization of cells almost doubled the production of fluorescence, indicating that diffusion into the cells of the rhodamine 110-based substrate is a limiting step in the accessibility of this substrate for intracellular proteases other than CD26/DPPIV. Therefore, we conclude that the nature of the fluorophore significantly affects interactions of these synthetic substrates with the active site of DPPIV as has been demonstrated before (
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Acknowledgments |
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We are grateful to Ms Trees Pierik and Mr Jan Peeterse for careful preparation of the manuscript and figures, respectively.
Received for publication December 23, 2002; accepted February 19, 2003.
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