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Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin.

Akimov SS, Krylov D, Fleischman LF, Belkin AM - J. Cell Biol. (2000)

Bottom Line: These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII.Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins.Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, American Red Cross, Rockville, Maryland 20855, USA.

ABSTRACT
The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifies adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading.

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Expression of exogenous tTG or its enzymatically inactive mutant tTG[C277→S], but not of FXIIIa promotes cell spreading. (A–E) REF52 cells were transfected with vector (vect.), wild-type tTG (tTG), tTG noncatalytic mutant C277→S (tTG[C277-S]), or FXIIIa (FXIIIa). (A) Expression levels of tTG on the surface of live transfectants were determined by immunostaining with 10 μg/ml polyclonal anti-tTG antibody and flow cytometry. (B) Expression levels of FXIIIa on the surface of live transfectants were determined by immunostaining with 10 μg/ml polyclonal antibody against FXIIIa and flow cytometry. (C) Transglutaminase activity in the cytosolic fractions of the transfectants was quantified by incorporation of [3H]putrescine into N,N-dimethylcaseine by transamidating enzymes present in cell lysates (Lorand et al. 1972). Shown are the results of triplicate determinations. (D) Transglutaminase activity on the surface of live transfectants was quantified by measuring cell-mediated incorporation of [3H]putrescine into N,N-dimethylcaseine. Bars represent the means of triplicate measurements. (E) Cells expressing vector, tTG, tTG[C277→S], or FXIIIa were photographed in regular culture. Bar, 20 μM.
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Figure 3: Expression of exogenous tTG or its enzymatically inactive mutant tTG[C277→S], but not of FXIIIa promotes cell spreading. (A–E) REF52 cells were transfected with vector (vect.), wild-type tTG (tTG), tTG noncatalytic mutant C277→S (tTG[C277-S]), or FXIIIa (FXIIIa). (A) Expression levels of tTG on the surface of live transfectants were determined by immunostaining with 10 μg/ml polyclonal anti-tTG antibody and flow cytometry. (B) Expression levels of FXIIIa on the surface of live transfectants were determined by immunostaining with 10 μg/ml polyclonal antibody against FXIIIa and flow cytometry. (C) Transglutaminase activity in the cytosolic fractions of the transfectants was quantified by incorporation of [3H]putrescine into N,N-dimethylcaseine by transamidating enzymes present in cell lysates (Lorand et al. 1972). Shown are the results of triplicate determinations. (D) Transglutaminase activity on the surface of live transfectants was quantified by measuring cell-mediated incorporation of [3H]putrescine into N,N-dimethylcaseine. Bars represent the means of triplicate measurements. (E) Cells expressing vector, tTG, tTG[C277→S], or FXIIIa were photographed in regular culture. Bar, 20 μM.

Mentions: To explore further the role of tTG in cell adhesion, rat REF52 fibroblasts were transfected with human tTG or its enzymatically inactive mutant C277→S. Flow cytometry of live REF52 transfectants was performed with a polyclonal anti-tTG antibody that recognizes both rat and human tTG. The cells expressing human tTG or tTG[C277→S] had increased amounts of tTG on the surface compared with vector controls, showing that both proteins were exported to the cell surface (Fig. 3 A). However, transfection of REF52 cells with human FXIIIa cDNA did not result in surface expression or secretion of this protein, indicating that mechanisms of externalization of these two transglutaminases are different (Fig. 3 B). Expression of tTG or FXIIIa, but not of the catalytic mutant tTG[C277→S], increased transglutaminase activity in the cytosolic fractions of the transfectants compared with vector-expressing cells (Fig. 3 C). Measurements of transglutaminase activity on the surface of live transfectants revealed a sharp increase after transfection with tTG, a reduction after transfection with tTG[C277→S], and no change in cells expressing FXIIIa (Fig. 3 D). In agreement with previous reports (Gentile et al. 1992; Verderio et al. 1998), overexpression of tTG strongly promoted cell spreading (Fig. 3 E). Interestingly, REF52 cells expressing tTG[C277→S] also appeared significantly more spread in regular culture than vector-transfected controls or FXIIIa transfectants (Fig. 3 E).


Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin.

Akimov SS, Krylov D, Fleischman LF, Belkin AM - J. Cell Biol. (2000)

Expression of exogenous tTG or its enzymatically inactive mutant tTG[C277→S], but not of FXIIIa promotes cell spreading. (A–E) REF52 cells were transfected with vector (vect.), wild-type tTG (tTG), tTG noncatalytic mutant C277→S (tTG[C277-S]), or FXIIIa (FXIIIa). (A) Expression levels of tTG on the surface of live transfectants were determined by immunostaining with 10 μg/ml polyclonal anti-tTG antibody and flow cytometry. (B) Expression levels of FXIIIa on the surface of live transfectants were determined by immunostaining with 10 μg/ml polyclonal antibody against FXIIIa and flow cytometry. (C) Transglutaminase activity in the cytosolic fractions of the transfectants was quantified by incorporation of [3H]putrescine into N,N-dimethylcaseine by transamidating enzymes present in cell lysates (Lorand et al. 1972). Shown are the results of triplicate determinations. (D) Transglutaminase activity on the surface of live transfectants was quantified by measuring cell-mediated incorporation of [3H]putrescine into N,N-dimethylcaseine. Bars represent the means of triplicate measurements. (E) Cells expressing vector, tTG, tTG[C277→S], or FXIIIa were photographed in regular culture. Bar, 20 μM.
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Figure 3: Expression of exogenous tTG or its enzymatically inactive mutant tTG[C277→S], but not of FXIIIa promotes cell spreading. (A–E) REF52 cells were transfected with vector (vect.), wild-type tTG (tTG), tTG noncatalytic mutant C277→S (tTG[C277-S]), or FXIIIa (FXIIIa). (A) Expression levels of tTG on the surface of live transfectants were determined by immunostaining with 10 μg/ml polyclonal anti-tTG antibody and flow cytometry. (B) Expression levels of FXIIIa on the surface of live transfectants were determined by immunostaining with 10 μg/ml polyclonal antibody against FXIIIa and flow cytometry. (C) Transglutaminase activity in the cytosolic fractions of the transfectants was quantified by incorporation of [3H]putrescine into N,N-dimethylcaseine by transamidating enzymes present in cell lysates (Lorand et al. 1972). Shown are the results of triplicate determinations. (D) Transglutaminase activity on the surface of live transfectants was quantified by measuring cell-mediated incorporation of [3H]putrescine into N,N-dimethylcaseine. Bars represent the means of triplicate measurements. (E) Cells expressing vector, tTG, tTG[C277→S], or FXIIIa were photographed in regular culture. Bar, 20 μM.
Mentions: To explore further the role of tTG in cell adhesion, rat REF52 fibroblasts were transfected with human tTG or its enzymatically inactive mutant C277→S. Flow cytometry of live REF52 transfectants was performed with a polyclonal anti-tTG antibody that recognizes both rat and human tTG. The cells expressing human tTG or tTG[C277→S] had increased amounts of tTG on the surface compared with vector controls, showing that both proteins were exported to the cell surface (Fig. 3 A). However, transfection of REF52 cells with human FXIIIa cDNA did not result in surface expression or secretion of this protein, indicating that mechanisms of externalization of these two transglutaminases are different (Fig. 3 B). Expression of tTG or FXIIIa, but not of the catalytic mutant tTG[C277→S], increased transglutaminase activity in the cytosolic fractions of the transfectants compared with vector-expressing cells (Fig. 3 C). Measurements of transglutaminase activity on the surface of live transfectants revealed a sharp increase after transfection with tTG, a reduction after transfection with tTG[C277→S], and no change in cells expressing FXIIIa (Fig. 3 D). In agreement with previous reports (Gentile et al. 1992; Verderio et al. 1998), overexpression of tTG strongly promoted cell spreading (Fig. 3 E). Interestingly, REF52 cells expressing tTG[C277→S] also appeared significantly more spread in regular culture than vector-transfected controls or FXIIIa transfectants (Fig. 3 E).

Bottom Line: These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII.Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins.Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, American Red Cross, Rockville, Maryland 20855, USA.

ABSTRACT
The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifies adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading.

Show MeSH
Related in: MedlinePlus