Anti-tissue transglutaminase antibodies from coeliac patients inhibit transglutaminase activity both in vitro and in situ - PubMed (original) (raw)

Anti-tissue transglutaminase antibodies from coeliac patients inhibit transglutaminase activity both in vitro and in situ

C Esposito et al. Gut. 2002 Aug.

Abstract

Background and aims: Coeliac disease (CD) is a multifactorial disorder which has an autoimmune component characterised by the occurrence of disease specific autoreactive antibodies against the enzyme tissue transglutaminase (tTG). The aim of this study was to investigate whether binding of antibodies to the enzyme influences tTG activity.

Methods: tTG activity was assayed in the presence of immunoglobulin A (IgA) and immunoglobulin G (IgG) purified from the serum of coeliac patients, CUB 7402 (an anti-tTG mouse monoclonal antibody), and human anti-tTG monoclonal antibodies derived from both intestinal lymphocytes from three patients with CD and from peripheral blood lymphocytes from healthy subjects. For our studies we used calcium treated and untreated recombinant human tTG. Furthermore, the effects of antibodies were determined by immunohistochemical detection of tTG activity in sections of human umbilical cord.

Results: IgG and IgA from CD patients inhibited tTG activity in vitro in a dose dependent manner, with a different rate of inhibition among patients. The monoclonal antibody CUB 7402 and human monoclonal antibodies displayed a dose dependent inhibitory effect towards the catalytic activity of the enzyme, both in vitro and in situ. Preincubation of tTG with CaCl(2) caused loss of the inhibitory effect due to CUB 7402 but not that caused by human monoclonal antibodies.

Conclusions: Purified CD IgA, IgG, as well as human anti-tTG monoclonal antibodies inhibited the enzymatic activity of human tTG both in vitro and in situ.

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Figures

Figure 1

Figure 1

Effect of purified serum IgG from coeliac patients (Nos 3 and 4) and from healthy controls on tissue transglutaminase (tTG) activity. Increasing amounts of IgG from patient No 4 (titre 24%), IgG from patient No 3 (titre 89%), and IgG from a pool of control patients (titre 2%) were incubated with 7 μg of proteins from Madin-Darby canine kidney (MDCK)-tTG cells at 37°C for one hour in a 100 μl final volume. The activity of tTG was calculated as a percentage of basal activity (no IgG addition) within MDCK-tTG cells. Data are presented as means (SD) from three separate experiments done in duplicate.

Figure 2

Figure 2

Effect of the monoclonal antibody anti-tissue transglutaminase (tTG) (CUB 7402) on tTG activity in response to Ca2+. Increasing concentrations of CUB 7402 were preincubated at 20°C for 10 minutes in 20 μl of 25 mM Tris/HCl, pH 8.0, in the absence or presence of 5 mM CaCl2, with 7 μg of proteins from Madin-Darby canine kidney (MDCK)-tTG cells. The samples were further incubated for one hour at 37°C in a 100 μl final volume. The activity of tTG was calculated as a percentage of basal activity (no CUB 7402 added) within MDCK-tTG cells. Data are presented as means (SD) from three separate experiments performed in duplicate.

Figure 3

Figure 3

Effect of single chain monoclonal antibodies on tissue transglutaminase (tTG) activity in response to Ca2+. Human monoclonal antibodies (0.5, 1.0, and 1.5 μg) were preincubated at 20° C for 10 minutes in 20 μl of 25 mM Tris/HCl, pH 8.0, in the absence (−) or presence (+) of 5 mM CaCl2, with 7 μg of proteins from Madin-Darby canine kidney (MDCK)-tTG cells. The samples were further incubated for one hour at 37°C in a 100 μl final volume. The activity of tTG was calculated as a percentage of basal activity (no antibodies added) within MDCK-tTG cells. Data are presented as means (SD) from three separate experiments performed in duplicate.

Figure 4

Figure 4

Effect of specific antibodies on tissue transglutaminase (tTG) activity in human umbilical cord. The signal corresponds to incorporation of substrate biotinylated monodansyl-cadaverine (A). Sections treated with the anti-tTG CUB 7402, diluted 1:25 (B), and with monoclonal antibody 4.2, undiluted (C), showed a significantly lower signal (original magnification ×400).

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