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Visualization of the moonlighting protein CD26DPPIV
Boonacker, E.
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Boonacker, E. (2003). Visualization of the moonlighting protein CD26DPPIV
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Download date: 18 Jun 2017
CHAPTER 4
Determination of reactions of enzymes and their kinetic parameters in
living cells by flow cytometry
Emil Boonacker and Cornells J.F. Van Noorden
Published in Nunez R, ed. Cytometry: Cytomics, proteomics, genomics.
Cytometry CD Vol 6. Multimedia Knowledge, Inc (www.mmke.com) in
conjunction with Purdue University Cytometry Labs, New York, 2002, pp 1-8
Determination of reactions of enzymes and their kinetic parameters in living cells by
flow cytometry
Emil Boonacker and Cornells J.F. Van Noorden
Academic Medical Center, University of Amsterdam, Department of Cell Biology and Histology.
Amsterdam. The Netherlands
Send correspondence to:
Prof.Dr. C.J.F. Van Noorden
Department of Cell Biology and Histology. Academic Medical Center
Meibcrgdreef 15. 1105 AZ Amsterdam. The Netherlands
phone: *31 20 566 4970; fax: *31 20 697 4156: e-mail: [email protected]
Quantitative enzyme histochemical methods are applied to determine enzyme reactions and kinetic
parameters of enzymes in intact cryostat sections of tissues and cell preparations to understand
how enzymes function in vivo [1]. Activity reflects an enzyme's physiological function and is
the ultimate outcome of regulation at transcriptional, translational and posttranslational levels.
However, these methods were traditionally applied to frozen and, thus, dead tissues and cells.
So, dynamic interactions between enzymes and macromolecular and structural elements as occur
in vivo are lost. These interactions are part of posttranslational control of the activity of enzymes
[2,3]. Therefore, we developed methods to analyze enzyme activity in living cells and used flow
cytometry for rapid measurements of the enzyme reactions in individual cells [4].
Since enzyme activities often display a heterogeneous character in cell populations, flow cytometry
is an ideal tool to study quantitatively enzyme activity in individual living cells. Furthermore,
activity can be related to other relevant parameters such as the amount of enzyme molecules
present as detected immunocytochemically, for example to determine posttranslational control
[5.6].
In contrast to fluorescence microscopy in which time lapse series of digital images can be made
of living cells during incubation to determine activity of a specific enzyme [4], each cell is
measured only once in flow cytometry. In the latter case, fading of fluorescence is of negligible
influence on the measurements, but on the other hand, enzyme reactions cannot be determined
in time per individual cell. We solved this problem by analysis of enzyme reactions in time in
large numbers of cells while the cells are incubated as shown in Fig. 1.
Proteases are a class of enzymes that play essential roles in health and disease, for example in the
turnover of extracellular matrix components, activation of the immune system, apoptosis. arthritis.
and metastasis of cancer. To visualize protease activity in individual living cells, a new class of
synthetic substrates containing the fluorescent group cresyl violet has been synthesized [4.7].
42
Cresyl violet has a different fluorescence spectrum when amino acids are attached, but that
changes after proteolytic liberation of the amino acids. To specifically detect liberated cresyl
violet, excitation at 591 nm and emission at 628 nm is needed [6].
The new synthetic substrates were used for the subcellular localization of cathepsin B activity in
cancer cells [7] and activity of dipeptidyl peptidase IV (CD26/DPPIV) in hepatocytes [4] and T
helper cells [6].
DPPIV is an ectopeptidase that is present on the plasma membrane of many different types of
cells. It is present on brush border membranes of intestine and kidney, where it is involved in the
digestion of polypeptides to provide substrates for peptide and amino acid transport systems [8].
Furthermore. DPPIV is homologous with CD26, which has a costimulatory function in T helper
cell activation. So, the CD26 molecule has at least 2 functions in T cells: a proteolytic function
that is involved in activation of procytokines or inactivation of cytokines and a signal transduction
function [9]. We investigated kinetic parameters of DPPIV/CD26 in individual living rat
hepatocytes and in polarized human T helper 1 and T helper 2 cells by incubating cells with
different concentrations of ala-pro-cresyl violet as substrate and analysis of the liberation of
cresyl violet by flow cytometry. In that way, we found that there is a strong and dynamic
posttranslational regulation of the kinetics of DPPIV in both cell types. This regulation strongly
determines its physiological function. For example, in rat hepatocytes the affinity of DPPIV is
high (low Km) when few enzyme molecules are present (low Vmax) and vice versa [4]. On the
other hand, T helper 1 cells contain 10-fold more CD26 molecules than T helper 2 cells but
DPPIV activity in the latter cells shows a Vmax and Km that are both 2-fold lower [6]. As a
result DPPIV activity in T helper 1 and T helper 2 cells at physiological substrate concentrations
is roughly the same despite the 10-fold difference in the number of enzyme molecules present
per cell. This phenomenon of posttranslational regulation becomes apparent only when activity
is analyzed in living cells.
Protocols for detection of DPPIV activity in living cells
A: Protocol for detection of DPPIV activity in living rat hepatocytes
Hepatocytes are isolated by collagenase perfusion of livers of Wistar rats as described by Caro el
al. [10]. Hepatocytes (5-10 mg dry mass/ml) are kept in Krebs-Henselheit bicarbonate medium
containing 1.3 mM Ca2', 10 mM Hcpes (pH 7.4), 20 mM glucose, and 1 mM octanoate on ice
until enzyme assays are performed. Prior to flow cytometric analysis, hepatocytes are stained
with the DNA dye, Hoechst 3.3.3.4.2 (1 ug/ml; Hoechst, Amsterdam, The Netherlands), for 30
min at 0°C. Hepatocytes are suspended 1:10 v/v in Krebs-Henselheit buffer.
Hepatocytes are mixed with a solution of the synthetic substrate, ala-pro-cresyl violet (Enzyme
Systems Products, Livermore CA, USA). A stock solution of 1 mM ala-pro-cresyl violet in
43
Ringer is made. Analysis is started by establishing forward scatter, and Hoechst and cresyl violet
fluorescence. Viable cells have a high forward scatter and low Hoechst fluorescence, whereas
cells with high fluorescence and low forward scatter are not likely to be intact or are loose nuclei
(Fig. 1). Viable hepatocytes are selected by gating the population with high forward scatter and
low Hoechst fluorescence (Fig. 1A).
The substrate is added in various concentrations (0-100 uM) to the cells in a tube and mixed
quickly and the sample differential pressure is boosted to prevent a lag fase in the kinetic
measurements. Then, the cells are analyzed for the amount of liberated cresyl violet per hepatocyte
in time (Fig. IB).
-I + J i l IVI'
FSC-HelflM
Fig. 1A. Flow cytometric analysis of Hoechst
33342 fluorescence (DNA dye) versus forward
scatter (FCS). A selection of intact living rat
hepatocytes was made on the basis of low
fluorescence and high forward scatter, indicated
by the oval window.
200
-«0
Tim*(5t2ü0s*e J
Fig. IB. Generation of cresyl violet fluorescence
by DPPIV activity as a function of time in rat
hepatocytes that were gated in Fig. IA with the
oval window, using 10 uM ala-pro-cresyl violet
as substrate, which was added at t=0 as indicated
by arrow.
B: Protocol for the simultaneous detection ot'CD26 expression and DPPIV
activity on living human T helper cells
Living T cells are harvested at different time points after stimulation and analysed for their
CD26 expression and DPPIV activity by means of flow cytometry. For CD26 detection, cells are
incubated for 30 min at 0°C with FITC-conjugated anti-human CD26 monoclonal antibody Tal
1 (1:80 dilution of a stock solution of 0.2 mg/ml) and washed twice in cold phosphate buffered
saline (PBS). pH 7.4. Cells were kept on ice prior to mixing with the enzyme incubation medium. Incubations were started at t=0 by suspending T cells in PBS containing 10 uM of the
DPPIV substrate ala-pro-cresyl violet (Enzyme Systems Products). Enzyme reactions are carried
out at 20°C.
44
Flow cytometric analysis of CD26 expression and formation of cresyl violet on living T helper
cells are performed on a FAC-star plus (Becton and Dickinson, Mountain View CA, USA),
using the software program CellQuest version 3.2. Analysis was started by establishing forward
scatter and then adding the substrate att=0. Analysis is performed at a rate of 200 cells/sec. The
parameters measured are time, forward scatter, side scatter, FITC fluorescence representing CD26
expression, and fluorescence of liberated cresyl violet formation (excitation at 591 nm and
emission at 628 nm). The increase in fluorescence is measured during 4 min (Fig. 2A) and
substrate specifity of DPPIV activity is tested in the presence of a specific inhibitor (Fig. 2B).
Fluorescence values are plotted against substrate concentrations. A hyperbolic curve is fitted to
the data with the use of a curve fitting program (Mac Curve Fit; Apple, Cupertino CA, USA) and
Vmax and Km values are calculated for living T helper cells according to [11].
TlITiB(£1ZDDJKj
Fig. 2A. Generation of cresyl violet fluorescence
by DPPIV activity on living human T helper I
cells, as a function of time, using 25 uM alapro-cresyl violet as synthetic substrate.
Time ( S i z m teci
Fig. 2B. Inhibition of DPPIV activity by
preincubation with 10 uM inhibitor P3420I
(Enzyme Systems Products) to demonstrate
specificity of DPPIV for the synthetic substrate
ala-pro-cresyl violet which was added in a
concentration of 25 uM.
References
1.
Van Noorden, C.J.F, and Jonges, G.N. Histochemical Journal 27:101-18 (1995).
2.
Swezey, R.R. and Epel, D. Journal of Cell Biology 103:1509-15 (1986).
3.
Rees B.B.. Swezey R.R.. Kibak K., and Epel D. Invertebrate Reproduction and
Development 30:123-34(1996).
4.
Van Noorden, C.J.F., Boonacker, E.. Bissell, E.R., Meijer, A.J., Van Marie, J. and Smith.
R.E. Analytical Biochemistry 252:71-7 (1997).
5.
Bleeker, F.E., Hazen, L.G.M.. Kohier, A., and Van Noorden, C.J.F Acta Histochemica.
102:247-57(2000).
6.
Boonacker et al, submitted
7.
Van Noorden, C.J.F., Jonges, G.N., Van Marie. J.. Bissell, E.R., Griffmi, P., Jans, M.,
Snel, J., and Smith, R.E. Clinical & Experimental Metastasis 16:159-17 (1998).
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Ganapathy, V., Pashley, D.H., Fonteles. M.C., and Leibach, F.H. Contributions to
Nephrology 42:10-8 (1984).
Dang. H.N.. Hafier. D.A.. Schlossman. S.F.. Breitmeyer. J.B. Journal of Immunology
125:42-57(1990).
Caro, L.H.P., Plomp, P.J.A.M.. Wolvetang. E.J. . Kerkhof. C , and Meijer. A.J. European
Journal of Biochemistry 175:325-29(1988).
Jonken A.. Geerts. W.. Charles. R., Lamers, W.H.. and Van Noorden. C.J.F.
Histochemistry and Cell Bioloay 106:437-43 (1996).