Fig 1.
Surface plasmon resonance (SPR) analysis of interactions between various DS/CS and TG2.
DS/CS chains that were biotinylated on their core protein remains were immobilized onto streptavidin-modified sensor chip and exposed to various concentrations of TG2 at a physiological ionic strength and 21°C, as described in Materials and methods. The dissociation phase of these interactions was generated by a rapid replacement of the TG2-containing solution with running buffer. (A) h CS/DS–sensorgrams obtained for the total human fibrotic fascia CS/DS; (B) h dec DS–sensorgrams obtained for human decorin DS from the fibrotic fascia; (C) h big DS–sensorgrams obtained for human biglycan DS from the fibrotic fascia; (D) p DS–sensorgrams obtained for porcine intestinal mucosa DS; (E) C-6-S–sensorgrams obtained for C-6-S from shark cartilage; (F) C-4-S–sensorgrams obtained for C-4-S from whale cartilage. Arrows show the association and dissociation phase of examined interactions. Asterisk shows signal reaction on a solution exchange.
Fig 2.
Typical reversed phase HPLC analysis of the porcine DS sulfation pattern.
The GAG was extensively depolymerized with chondroitinase ABC, and the released unsaturated disaccharides were labeled with fluorophore 2-aminoacridone and separated by HPLC, as described in Materials and methods. The elution positions of individual disaccharides are indicated. The percentage content of individual disaccharides is as follow: Δdi2,4S – 1.4±0.2%; Δdi4,6S – 3.7±0.2%; Δdi4S – 88.4±0.1%; Δdi6S – 5.7±0.6%; Δdi0S 0.8±0.3% (mean ± S.D. of five independent analyses).
Table 1.
Kinetic parameters characterizing the interactions of transglutaminase 2 (TG2) with structurally different types of dermatan sulfate (DS)/ chondroitin sulfate (CS).
Fig 3.
Typical saturation binding of TG2 to the immobilized DS.
Incubation of the DS with an increasing amounts of TG2 was conducted, as described in Materials and methods. The analysis was carried out in the form of three independent experiments performed in duplicate. Data that are shown represent results (the mean ± S.D.) of one of them. The inset shows a Scatchard-type plot of the experimental data for the porcine DS binding to TG2.
Fig 4.
The competition of heparin for the interaction between the DS and TG2.
The DS that was immobilized onto sensor disc was exposed to 150 nM of TG2 without heparin (control) or with indicated amounts of heparin, as described in Materials and methods. (A) Representative sensorgrams that show the interaction between the DS and TG2 in the presence of heparin. (B) The interference of heparin in the DS-TG2 interaction was estimated as reduction in the surface plasmon resonance (SPR) signal. The results (the observed SPR response) are means ± S.D. of at least 3 independent experiments. a–statistically significant differences compared to the binding without heparin (p ≤ 0.05).
Fig 5.
TG2 is associated to DS/CS side chains of decorin molecules secreted into the culture medium by human keloid fibroblasts.
(A) Molecular complexes that contained native decorin or decorin treated with chondroitinase ABC (Chase ABC) to eliminate DS/CS chain were immunoprecipitated (IP) with an anti-human decorin core protein antibody (anti-dec) from the equal portions of the conditioned medium (each portion contained 5 μg of GAG) and separated by reducing SDS-PAGE followed by Western blot detection with the anti-dec or an anti-human TG2 serum. Lane 1 –the anti-dec antibody applied as negative control; lane 2 –the core protein of decorin; lane 3 –native decorin; lane 4 –components that were co-immunoprecipitated with the decorin core protein and detected by the anti-TG2 antibody; lane 5 –molecules that were co-immunoprecipitated with native decorin and detected by the anti-TG2 antibody; lane 6 –the cross-reactivity between the antibody that was used for the co-immunoprecipitation (mouse anti-decorin) and the sera were applied for the Western blotting (rabbit anti-human Tg2 and goat anti-rabbit IgG). The migration position of mass markers is shown. (B) The equal portions of the conditioned medium (each containing 5 μg of GAG) from the keloid fibroblasts, which have been cultured in the presence of 50 μM putrescine, were untreated or digested with Chase ABC and subsequently incubated with a human recombinant TG2, as described in Materials and methods. Molecular complexes that contained decorin or its core protein were immunoprecipitated and subjected to Western blot analysis firstly for TG2, and after stripping for decorin. Lane 1 –TG2 that co-immunoprecipitated with the untreated decorin; lane 2 –TG2 that co-immunoprecipitated with Chase ABC treated decorin; lane 3 –decorin untreated with Chase ABC; lane 4 –decorin digested with this enzyme. The migration position of the human recombinant TG2 (hrTG2) is indicated by arrowhead. All the experiments were performed in triplicate.
Fig 6.
Acceptors of TG2 in the extracellular matrix of human fascia.
(A) The protein was extracted from tissue samples that were treated with chondroitinase ABC (Chase ABC) or heparinase III (Hep) to eliminate DS/CS or HS, respectively. Components that were extracted from 1 mg of the dry tissue were subjected to reducing SDS-PAGE and Western blotting using the anti-human TG2 antibody, as described in Materials and methods. Lane 1 –an extraction with buffer, pH 8.0; lane 2 –an extraction with buffer, pH 8.0 and Chase ABC; lane 3 –an extraction with buffer, pH 7.5 and heparinase III; lane 4 –an extraction with buffer, pH 7.5. Arrow indicates the migration position of TG2. Relative quantification of the free TG2 extractability is presented as means ± S.D. of at least three independent experiments. a–differences statistically significant versus control (buffer treated tissue samples) (p ≤ 0.05). (B) TG2 that was extracted from 1 mg of the dry human fascia after Chase ABC-dependent degradation of tissue DS/CS in the presence of anti-fibronectin (anti-FN) and/or anti-collagen I (anti-Col I) antibodies was separated by reducing SDS-PAGE followed by Western blotting with the anti-human TG2 antibody. Lane 1 –an extraction with anti-fibronectin and Chase ABC; lane 2 –control extraction with anti- fibronectin; lane 3 –an extraction with anti-collagen I and Chase ABC; lane 4 –control extraction with anti-collagen I; lane 5 –an extraction with anti-fibronectin, anti-collagen I and Chase ABC; lane 6 –control extraction with anti-fibronectin and anti-collagen I. Arrow indicates the migration position of TG2. Relative quantification of the free TG2 extractability is presented as means ± S.D. of five independent experiments. a–differences statistically significant versus control (tissue samples treated only with proper antibody and buffer) (p ≤ 0.05).
Fig 7.
The GAG effect on TG2-dependent transamidating activity.
The transamidating activity of the enzyme was estimated as a quantity of biotinylated cadaverine incorporated to CBZ-Gln-Gly (A) or collagen type I (B) without GAGs (control) or with indicated concentrations (μg/ml) of the porcine DS (p DS), the total DS/CS from the fibrotic fascia (h DS/CS), shark cartilage C-6-S or heparin as described under Materials and methods. Relative TG2 activity is expressed as a percentage of the control. The results are means ± S.D. of at least 3 independent experiments. a–differences statistically significant versus control (p ≤ 0.05).