Heparan sulfate proteoglycans are receptors for the cell-surface trafficking and biological activity of transglutaminase-2

Abstract

Transglutaminase type 2 (TG2) is both a protein cross-linking enzyme and a cell adhesion molecule with an elusive unconventional secretion pathway. In normal conditions, TG2-mediated modification of the extracellular matrix modulates cell motility, proliferation and tissue repair, but under continuous cell insult, higher expression and elevated extracellular trafficking of TG2 contribute to the pathogenesis of tissue scarring. In search of TG2 ligands that could contribute to its regulation, we characterized the affinity of TG2 for heparan sulfate (HS) and heparin, an analogue of the chains of HS proteoglycans (HSPGs). By using heparin/HS solid-binding assays and surface plasmon resonance we showed that purified TG2 has high affinity for heparin/HS, comparable to that for fibronectin, and that cell-surface TG2 interacts with heparin/HS. We demonstrated that cell-surface TG2 directly associates with the HS chains of syndecan-4 without the mediation of fibronectin, which has affinity for both syndecan-4 and TG2. Functional inhibition of the cell-surface HS chains of wild-type and syndecan-4-null fibroblasts revealed that the extracellular cross-linking activity of TG2 depends on the HS of HSPG and that syndecan-4 plays a major but not exclusive role. We found that heparin binding did not alter TG2 activity per se. Conversely, fibroblasts deprived of syndecan-4 were unable to effectively externalize TG2, resulting in its cytosolic accumulation. We propose that the membrane trafficking of TG2, and hence its extracellular activity, is linked to TG2 binding to cell-surface HSPG.

Figures

FIGURE 1.

TG2 binds immobilized heparin and HS. A, the binding of purified TG2 to low molecular weight heparin was performed over 2 h at 37 °C in the presence of 2 mm EDTA, 2 mm CaCl2, or 2 mm EDTA plus 100 μm GTPγS, as described under “Experimental Procedures.” The mean ± S.D. of three independent experiments performed in triplicate is shown. B, binding assays to immobilized FN and BSA were used as positive and negative binding controls, respectively. C, binding of purified TG2 to immobilized HS was performed as described in A, in the presence of 2 mm EDTA. D, affinity of TG2 for heparin calculated by surface plasmon resonance. TG2 was injected over a heparin activated surface for 8 min at a flow rate of 10 μl/min, after which running buffer was injected, and the response in RU was recorded as a function of time. Sensorgrams were obtained with increasing TG2 concentrations, and analyzed with the Biaeval 3.1 software.

FIGURE 2.

Adhesion of wild-type (TG2+/+) and TG2-null (TG2−/−) MEF to immobilized heparin, HS, and control BSA. Serum-starved cells were allowed to adhere for 1 h in serum-free medium, after that the attached cells were fixed, stained with crystal violet and quantified as described under “Experimental Procedures.” Values are the mean ± S.D. of a representative experiment performed in triplicate; data were normalized considering attachment of TG2+/+ on FN as 100. ***, statistically significant differences in adhesion (p < 0.001). The bars represent 20 μm.

FIGURE 3.

Association of TG2 to syndecan-4. A, membrane lysates from wild-type and TG2-transfected HOB (HOB-TG14) were immunoprecipitated with anti-syndecan-4 antibody (S4) and, as negative controls, anti-gliadin antibody (Gl) or protein G-Agarose only (B). Immunoprecipitates were subjected to Western blot analysis for TG2 and syndecan-4. Arrowhead indicates TG2; asterisk, glycanated S4 (∼200 kDa); Hc antibody heavy chain (∼50 kDa). B, the same membrane lysates were immunoprecipitated with anti-TG2 antibody, digested or not with heparitinase (Hep) and subjected to Western blot analysis for syndecan-4 and TG2. Arrow, S4 core protein dimer. C, TG2 and syndecan-4 co-localization in HOB. TG2 was detected by mouse anti TG2 antibody and syndecan-4 with rabbit anti syndecan-4 antibody followed by sheep anti-mouse IgG FITC and donkey anti-rabbit IgG AlexaFluor568. Two separate fields are shown. Co-localization is pointed by the arrows. The bar indicates 20 μm. D, flow cytometry of live HOB stained with a FITC-conjugated monoclonal anti-HS chains antibody, before and after heparitinase (Hep) digestion of HS. The arrow indicates the shift of mean fluorescence. E, the syndecan-4 immunoprecipitations described in A were repeated using membrane preparations from cells preincubated or not with heparitinase (Hep) prior to immunoprecipitation. TG2 indicates 200 ng of standard purified TG2. Three replicate blots are shown, and quantification of syndecan-4 co-immunoprecipitated TG2 is presented in the histogram. Arrowheads point to TG2 co-immunoprecipitated with syndecan-4. Values represent mean intensity relative to that of TG2 standard ± S.D. *, p < 0.05; **, p < 0.01. F, the syndecan-4 immunoprecipitations described in A were conducted in the presence of peptide P3 that mimics the FN binding site within TG2. Arrowheads indicate TG2 or FN co-immunoprecipitated with syndecan-4. G, inhibition of TG2-FN binding, assayed using 20 nm and 50 nm purified TG2 on FN in the presence of increasing concentrations of peptide P3. The experiments were repeated at least three times.

FIGURE 4.

Cell-surface HS affect extracellular TG2 activity of primary dermal fibroblasts adherent to FN. A, cell-surface TG2 activity was measured in wild-type (S4+/+), syndecan-4-null (S4−/−), and syndecan-4-null MDF with added-back S4 by cell transfection of pcDNAhS4. In some instances, cells were either pretreated with heparitinase (Hep) or with the HS antagonist surfen (12 μm) or incubated with medium only, for 1 h at 37 °C. Afterward cells were seeded on FN and assayed for TG2 activity in the presence of Hep or surfen. The cell-surface TG2 activity was expressed as normalized mean absorbance at 450 nm ± S.D., using wild-type untreated samples as normalizers (typical optical density = 0.6). **, statistically significant decrease in cell-surface TG2 activity (p < 0.01). B, the expression of TG2 in wild-type and syndecan-4-null MDF was analyzed by reverse transcription-PCR, using triplicate samples of total RNA/cell type, followed by quantification of bands intensities by densitometry. Values (TG2 expression) are mean ratios of TG2 intensity versus control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The expression of total TG2 in cell homogenates was evaluated by means of a TG2 activity plate assay (Total TG2 activity), as described under “Experimental Procedures.” TG2 activity data represent mean values ± S.D. of three separate experiments, using values of wild-type cells (typical optical density = 0.5) as normalizers. TG2 expression was also analyzed by Western blotting of total cell homogenates using polyclonal anti-TG2 antibody Ab10445/50. C, re-expression of syndecan-4 (hS4) in syndecan-null MDF was tested by reverse transcription-PCR on RNA isolated 48 h after transfection of pcDNAhS4 or the empty vector pcDNA. Endogenous mouse syndecan-4 (mS4) was detected in wild-type MDF (S4+/+). Expression of glyceraldehyde-3-phosphate dehydrogenase was monitored to confirm equal loading of RNA. MW, molecular DNA markers (1-kb Promega). D, S4+/+ and S4-null MDF were allowed to attach to FN-coated wells, in the same conditions as in the cell-surface TG2 assay described in A (in serum-free conditions), but in the absence of amine substrate. Where indicated, cells had been incubated with 6 and 12 μm Surfen. At the end of the 2-h incubation, cell attachment was quantified as described under “Experimental Procedures.” The bars indicate 20 μm.

FIGURE 5.

Visualization of in situ TG2 activity in wild-type and syndecan-4-null dermal fibroblasts. In situ TG2 activity of S4+/+ and S4-null MDF was visualized by the incorporation of fluorescein-cadaverine into endogenous substrates. R-283, a site-specific inhibitor of TG activity was used as a control at 200 μm. Nuclei were stained with propidium iodide to facilitate cell counting. The average fluorescence produced by fluorescein excitation was quantified by Leica TCSNT image processing in five random fields and expressed as mean fluorescence/cell. *, significantly different TG2 activity (p < 0.05). The bars correspond to 40 μm.

FIGURE 6.

Heparin binding does not affect the cross-linking activity of purified TG2. The cross-linking activity of purified TG2 was assayed in the absence and presence of heparin as described under “Experimental Procedures.” Data are the mean values ±S.D. of a representative experiment undertaken in triplicate. p > 0.05.

FIGURE 7.

Importance of syndecan-4 in cell-surface TG2 location. A, S4+/+ and S4-null MDF were fixed, permeabilized, and stained with monoclonal anti-TG2 antibody (Cub7402), which was revealed by using a FITC-conjugated secondary antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Specimens were imaged using fluorescence or both phase contrast and fluorescence illumination in combination. The arrows indicate TG2 localization at the cell periphery. The bars correspond to 20 μm. B, TG2 staining was performed as described in A; actin stress fibers were stained using TRITC-phalloidin. Arrowheads indicate TG2 localization at cell-matrix adhesions; small arrows indicate TG2 released in the ECM. The bars correspond to 20 μm. C, TG2 and syndecan-4 were co-immunodetected using Cub7402 and anti-polyclonal anti-syndecan-4 antibody, which were revealed by anti-mouse IgG-Rhodamine RedX and anti-rabbit IgG-Alexafluor488, respectively. Nuclei were stained with 4′,6-diamidino-2-phenylindole. The arrows point at the cell-matrix adhesion sites where TG2 and syndecan-4 co-localize. The bars indicate 20 μm. D, for detection of matrix-associated TG2, S4+/+ and S4-null MDF were cultured in the presence of Cub7402 before fixation and incubation with FITC-conjugated secondary antibody. Average signal of matrix TG2 per single cell was quantified in three separate fields and is displayed in the graph. Values are normalized considering S4+/+ signal as 100. ** indicates p < 0.01. E, analysis of the cellular distribution of TG2 in S4+/+ and S4-null MDF. Cells were fractionated into membrane (M) and cytosolic (C) fractions, as described under “Experimental Procedures.” The level of TG2 was then assessed by Western blotting using anti-TG2 antibody and equal loading was verified on a parallel blot of membrane and cytosolic fractions, respectively, immunoprobed with anti-Na/K ATPase antibody (membrane marker) and anti-tubulin antibody. F, S4+/+ and S4-null MDF were fractionated and the total TG2 activity assayed as described under “Experimental Procedures.” The asterisks indicate a significant difference between S4+/+ and S4-null MDF membrane and cytosolic TG2 (p < 0.02). There was no significant difference in the total level of TG2 between the two cell types.