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First published online 21 August 2007
doi: 10.1242/jcs.010397

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Cell-surface transglutaminase undergoes internalization and lysosomal degradation: an essential role for LRP1

¹ Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
² Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, MD 21201, USA
³ Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
⁴ Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, USA
⁵ Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD 21201, USA

View larger version (23K): [in this window] [in a new window]   Fig. 1. tTG is internalized from the cell surface by means of a cholesterol- and dynamin-dependent mechanism. (A) Endocytosis of tTG from the surface of WI-38 fibroblasts. The Fab fragments of mouse mAb 4G3 against tTG were incubated with WI-38 fibroblasts at 4°C. Next, the cells were warmed up to 37°C for 15 or 60 minutes and the Fab fragments remaining on the cell surface were stripped by a low-pH treatment. The internalized tTG–4G3-Fab complexes were detected by immunofluorescence after cell permeabilization. (B) Plasma membrane cholesterol is required for internalization of cell-surface tTG. NIH3T3 fibroblasts expressing exogenous tTG were treated with 10 mM M--CD and then incubated for 3 hours at 37°C in DMEM-FBS without the inhibitor. Cell-surface proteins were biotinylated with membrane-impermeable sulfo-NHS-LC-biotin. Biotinylated proteins were isolated and total cellular and cell-surface fractions were analyzed for tTG by SDS-PAGE and immunoblotting. The numbers beneath the tTG bands display relative intensities compared with a value of 1.0 assigned to untreated cells. Shown is a representative result of three independent experiments. (C) GTPase activity of dynamin-2 is required for endocytosis of cell-surface tTG. MRC-5 fibroblasts were transiently transfected with either wild-type (wt) or a GTPase-deficient dynamin-2 mutant (K44A) with a hemagglutinin (HA) tag. 40 hours after transfection, antibody-uptake assays with the Fab fragments of mAb 4G3 were performed for 15 minutes as described in the legend to panel 1A. After permeabilization, the cells were co-stained for the transfected dynamin-2 or the dynamin-2 K44A mutant with antibody against HA (left panels) and for the tTG–4G3-Fab complexes (right panels). Arrows mark multiple vesicles containing the internalized tTG–4G3-Fab complexes in the cells expressing the endogenous or transfected wild-type dynamin-2, but not its (K44A) mutant. The percentages of cells with positive staining for the internalized tTG–4G3-Fab complexes (mean pixel intensity within the cell area 30) were calculated for the populations of untransfected and wild-type dynamin- or mutant K44A-dynamin-transfected MRC-5 fibroblasts (n=120, lower panel).

View larger version (83K): [in this window] [in a new window]   Fig. 2. The internalized cell-surface tTG is transported through endosomal compartments to lysosomes. Antibody-uptake experiments were performed as described above in Fig. 1A. Double-immunofluorescence staining of WI-38 fibroblasts for internalized tTG–4G3-Fab complexes (central panels) and endocytic markers (left panels), including EEA1 (early endosomes, 10 minutes of endocytosis), Rab7 (late endosomes, 30 minutes of endocytosis), Arf1 (ECV/Golgi, 30 minutes of endocytosis) or Lamp-1 (lysosomes, 120 minutes of endocytosis). Merged images are shown in the right panels. Arrows indicate colocalization of the internalized tTG–4G3-Fab complexes with various organelle markers.

View larger version (47K): [in this window] [in a new window]   Fig. 3. The internalized cell-surface tTG undergoes proteolytic degradation. (A) tTG colocalizes with its binding partner, the 1 integrin subunit, early after endocytosis from the cell surface. Antibody-uptake experiments were performed for 15 minutes, as described above in Fig. 1A, with WI-38 fibroblasts and the Fab fragments of mouse mAb 4G3 against tTG and rat mAb 9EG7 against 1 integrins. (B) Cell-surface tTG, but not the 1 integrin subunit, is degraded after internalization. Surface biotinylation and endocytosis assays were performed with CHO cells and membrane-impermeable SH-cleavable sulfo-NHS-SS-biotin. Biotinylated proteins were isolated and tTG and 1 integrin were detected by SDS-PAGE and immunoblotting in the fraction of proteins internalized from the cell surface. The intensities of protein bands at various time points of internalization were quantified by densitometry and compared with those of cell-surface tTG and 1 integrins before endocytosis. Shown are the means ± s.d. for three independent experiments. Asterisks indicate proteolytic fragments of tTG.

View larger version (58K): [in this window] [in a new window]   Fig. 4. Cell-surface tTG is internalized by means of clathrin- and lipid-raft-(caveolae-) dependent pathways. (A) Cell-surface tTG colocalizes with transferrin and clathrin during internalization. Antibody-uptake assays were performed with WI-38 fibroblasts as described above in Fig. 1A with the Fab fragments of mouse mAb 4G3 against tTG and FITC-labeled transferrin (upper panels), or the anti-tTG Fab fragments only, and the cells were co-stained for clathrin after 15 minutes of internalization (lower panels). (B) Internalization of cell-surface tTG is attenuated by the inhibitors of clathrin- and caveolae-dependent endocytosis. WI-38 fibroblasts were left (c) untreated or were treated with (h) hyperosmotic 0.45 M sucrose or (f) 5 µg/ml filipin before surface biotinylation with membrane-impermeable SH-cleavable sulfo-NHS-SS-biotin. (C) Co-distribution of tTG with caveolin-1 and 1 integrins on the cell surface. Live non-permeabilized MRC-5 fibroblasts were triple-labeled with antibodies against the three proteins. Arrows mark the sites of colocalization of tTG and caveolin-1 on the cell surface. (D) Inhibition of caveolae-dependent endocytosis increases cell-surface levels of tTG. NIH3T3 fibroblasts expressing tTG were either left untreated or were treated with filipin. Surface labeling of untreated and filipin-treated cells with sulfo-NHS-LC-biotin was followed by isolation of biotinylated (cell surface) tTG. (E) Downregulation of caveolin-1 promotes endocytosis of cell-surface tTG. Surface biotinylation and endocytosis assays were performed with U-251 glioma cells expressing vector only or caveolin-1 siRNA, using membrane-impermeable SH-cleavable sulfo-NHS-SS-biotin. Biotinylated proteins in (B,E) were isolated and tTG was detected by SDS-PAGE and immunoblotting in the fraction of proteins internalized from the cell surface. The numbers beneath the tTG bands show relative intensities compared with the value of 1.0 assigned to untreated cells (D) or cells before internalization (B,E). Shown in (B,D,E) are representative results from three independent experiments.

View larger version (60K): [in this window] [in a new window]   Fig. 5. PDGF and fibronectin promote endocytosis of cell-surface tTG. (A) PDGF accelerates internalization and degradation of cell-surface tTG. Quiescent serum-starved WI-38 fibroblasts surface-labeled with sulfo-NHS-SS-biotin, were either left untreated [`C'], or were treated with (c) PDGF only, or with (h) PDGF and the inhibitors of clathrin-dependent (0.45 M sucrose) or (f) caveolae-dependent (5 µg/ml filipin) endocytosis. (B) The internalization of tTG from the cell surface is stimulated by fibronectin. Fibronectin-null mouse embryonic fibroblasts expressing exogenous tTG were grown in fibronectin-depleted 10% serum with or without 50 µg/ml fibronectin (Fn) and were surface-biotinylated using sulfo-NHS-SS-biotin. Biotinylated proteins were isolated and tTG was detected by SDS-PAGE and immunoblotting in the fraction of proteins internalized from the cell surface (A,B). Proteolytic fragments of tTG are marked with asterisks. Shown in (A,B) are representative results of three independent experiments.

View larger version (36K): [in this window] [in a new window]   Fig. 6. tTG is colocalized with LRP1 during internalization and the two interact on the cell surface. (A) tTG colocalizes with LRP1 early after endocytosis from the cell surface. Antibody-uptake experiments were performed for 5 or 15 minutes with WI-38 fibroblasts and the Fab fragments of mouse mAb 4G3 against tTG and rabbit antibody Rb2629 against LRP1, as described above in Fig. 1A. The internalized tTG–4G3-Fab and LRP1–anti-LRP1 complexes were detected by immunofluorescence after cell permeabilization. Note the significant colocalization of the internalized proteins in the numerous peripheral endocytic vesicles. (B) tTG is associated with LRP1. Extracts of quiescent (–) and PDGF-treated (+) NIH3T3 cells expressing exogenous tTG were subjected to immunoprecipitation with non-immune IgG or antibody 2629 against LRP1. The resulting immune complexes were examined by SDS-PAGE and immunoblotting for tTG and LRP1. (C) tTG enhances the association of LRP1 with 1 integrins. 1 integrins were immunoprecipitated from the extracts of quiescent (–) or PDGF-treated (+) NIH3T3 cells lacking (vector) or expressing tTG. The resulting immune complexes were analyzed for LRP1, tTG and 1 integrins by SDS-PAGE and immunoblotting. (D) Cell-surface tTG mediates a shift of LRP1 towards high-density membrane or ECM fractions enriched in tTG and fibronectin. NIH3T3 fibroblasts lacking (vector) or expressing tTG were surface-biotinylated with membrane-impermeable sulfo-NHS-LC-biotin. Carbonate extracts at pH 11 of the cells were subjected to ultracentrifugation in a discontinuous (45%-35%-5%) sucrose gradient. Biotinylated (cell surface) proteins from each gradient fraction were isolated and analyzed by SDS-PAGE and immunoblotting with antibodies against LRP1, tTG and fibronectin. The high-density fractions enriched in tTG and fibronectin and expressing increased amounts of LRP1 in the tTG-expressing cells are marked with asterisks. The numbers beneath the LRP1 bands show relative intensities compared with the value of 1.0 assigned to cells lacking tTG in the absence of PDGF. Shown are representative results of three independent experiments.

View larger version (49K): [in this window] [in a new window]   Fig. 7. tTG interacts directly with LRP1 by means of the catalytic domain. (A) The interaction of purified LRP1 with tTG immobilized on plastic wells. Binding assays were performed as described in Materials and Methods in the presence or absence of the LRP1 interactor RAP. Immobilized BSA and RAP were used as negative and positive binding controls, respectively. (B) The interaction of purified tTG with LRP1 and soluble VLDL receptor (sVLDLR) immobilized on plastic wells. Binding assays were performed as described in Materials and Methods with BSA used as a negative binding control. Shown in (A,B) are the means ± s.d. for two independent experiments performed in triplicate. (C) LRP1 interacts with the second (catalytic) domain of tTG. Full-length tTG and its deletion mutants (Hang et al., 2005), all containing the C-terminal c-Myc tag, were expressed in NIH3T3 fibroblasts. Cell extracts were analyzed by SDS-PAGE and immunoblotting with antibody against c-Myc for total expression levels of full-length tTG and its deletion mutants (upper panel). LRP1 was immunoprecipitated from cell extracts with the rabbit antibody Rb2629, and the resulting LRP1 immune complexes were analyzed by SDS-PAGE and immunoblotting with antibody against c-Myc (lower panel). Molecular mass markers (kDa) are shown to the right of the gels.

View larger version (69K): [in this window] [in a new window]   Fig. 8. Internalization of cell-surface tTG requires the endocytic receptor LRP1. LRP1 deficiency inhibits internalization of cell-surface tTG. Wild-type (CHO wt) and LRP1-deficient (CHO 13-5-1) CHO cells (A) or normal (MEF) and Lrp1–/– (PEA-13) mouse embryonic fibroblasts expressing exogenous tTG (B) were surface-biotinylated with sulfo-NHS-SS-biotin and subjected to internalization assays. (A,B) Biotinylated proteins were isolated and tTG was detected by SDS-PAGE and immunoblotting in the fraction of proteins internalized from the cell surface. Proteolytic fragments of tTG are marked with asterisks. Graphs on the right show the levels of internalized tTG for each cell type at different time points of internalization. The values presented are expressed as percentages of those obtained for cell-surface tTG before internalization. The results are representative of three independent experiments performed for each cell type (means ± s.d.).

View larger version (42K): [in this window] [in a new window]   Fig. 9. LRP1 deficiency and impairment of endocytosis increase extracellular transglutaminase activity and cell adhesion by upregulation of cell-surface tTG. Surface tTG levels (A), extracellular transglutaminase activity (B) and adhesion to the 42-kDa fragment of fibronectin (C) were determined for normal (MEF) and Lrp1–/– (PEA-13) mouse embryonic fibroblasts lacking or expressing tTG (left panels) and for wild-type CHO cells, either untreated or treated with 2 µM bafilomycin or 35 µM chloroquine for 16 hours (right panels). (A) LRP1 deficiency and inhibition of endocytosis upregulate the levels of cell-surface tTG. The steady-state surface tTG levels were determined by surface labeling with sulfo-NHS-LC-biotin, isolation of biotinylated proteins, SDS-PAGE and immunoblotting, and expressed as percentages of the total tTG levels. The results are representative of three independent experiments performed for each cell type (means ± s.d.). (B) The absence of LRP1 and ablation of endocytosis increase the enzymatic activity of tTG on the cell surface. Cell-mediated incorporation of 3H-putrescine into N,N-dimethylcaseine was measured as described previously (Belkin et al., 2001). (C) The lack of LRP1 and treatment with endocytic inhibitors enhance adhesion of cells to the tTG-binding 42-kDa fragment of fibronectin (Radek et al., 1993). Adhesion to the immobilized 42-kDa fragment was measured as described previously (Akimov et al., 2000). The data in (B,C) are means of two independent experiments performed in triplicate.

View larger version (51K): [in this window] [in a new window]   Fig. 10. A scheme summarizing the regulation of tTG functions on the cell surface by LRP1-mediated endocytosis. In LRP1-expressing cells with active endocytosis, the receptor downregulates surface tTG by internalization and targeting it to lysosomes for degradation, thereby decreasing cell-matrix adhesion and ECM crosslinking. In cells with no LRP1 or with impaired endocytosis, tTG accumulates on the surface owing to lack of internalization. For further information, see main text.