The interaction of angiocidin with tissue transglutaminase

Abstract

Angiocidin, a matrix bound and tumor associated protein, has been shown to inhibit tumor progression and angiogenesis. We previously demonstrated that angiocidin binds to thrombospondin-1 and alpha2beta1 integrin. We now show that angiocidin binds and is a preferred substrate for tissue transglutaminase-2 (tTgase). Angiocidin bound tTgase saturably with a Kd of 26 nM, while an angiocidin deletion mutant missing the matrix binding domain of angiocidin failed to bind tTgase. tTgase colocalized with angiocidin on endothelial cells. tTgase bound anti-angiocidin immunoprecipitates of endothelial cell lysates. Breast cancer cells expressing high levels of tTgase attached to angiocidin immobilized on tissue culture plates. Angiocidin was a preferred substrate for tTgase forming high molecular weight cross-linked multimers when treated with tTgase. Cross-linked angiocidin contained iso-peptide bonds as demonstrated by Western blotting and immunohistochemical colocalization studies using endothelial cells treated with angiocidin. Cross-linked angiocidin inhibited cell migration in contrast to monomeric angiocidin and inhibited localization of fibronectin (FN), a pro-tumorigenic matrix protein, into the extracellular matrix (ECM) of tumor and HUVE cells. Our studies provide an additional explanation for the anti-tumor activity of angiocidin suggesting that cross-linked angiocidin disrupts the tumor ECM making it less permissive for tumor growth.

Fig. 1

The interaction of angiocidin with tTgase. A- Direct binding of angiocidin to immobilized tTgase; B- tTgase Western blot of anti-angiocidin HUVE cell immunoprecipitates; C – Immunohistochemical localization of tTgase and his-tag angiocidin in cultured HUVE cells; D- Adhesion to angiocidin of MDA-MB-231 breast expressing low, medium, and high levels of tTgase. Low expressors are designated Low, medium expressors are designated wildtype (WT) and high expressors are designated High. The lower panel in D shows the cell lines Western blotted for tTgase. All experiments were repeated three times to establish reproducibility. The experiments shown are representative of three experiments performed for each panel.

Fig. 2

tTgase-mediated cross-linking of angiocidin and matrix proteins. A- left panel monomeric and cross-linked angiocidin analyzed by SDS-PAGE, right panel- Anti-isopeptide bond blot of monomeric and cross-linked angiocidin. B- SDS-gels of angiocidin, TSP-1, FN, and laminin treated with tTgase for various times. All experiments were repeated three times to establish reproducibility. The experiments shown are representative of three experiments performed for each panel.

Fig. 3

Angiocidin sequence requirements for tTgase-mediated cross-linking of angiocidinA-Comparison of tTgase mediated cross-linking of angiocidin and two deletion mutants of angiocidin, M1 and M2 as assessed by SDS-PAGE B-The effect of M1 peptide on tTgase mediated cross-linking of angiocidin as assessed by SDS-PAGE. All experiments were repeated three times to establish reproducibility. The experiments shown are representative of three experiments performed for each panel.

Fig. 4

The effect of cross-linked angiocidin on HUVE cell migration. The migration through of HUVE cells through collagen, cross-linked angiocidin plus collagen or as a control in the presence of tTgase was measured as described in Materials and methods. All experiments were repeated three times to establish reproducibility. The experiments shown are representative of three experiments performed.

Fig. 5

The co-localization of angiocidin and isopeptide bond formation in HUVE cells treated with angiocidin. A- Immunohisto-localization of isopeptide bond formation in HUVE cells treated with different concentrations angiocidin B-Co- immunohisto-localization of isopeptide bond formation and his-tagged angiocidin in HUVE cells treated with his-tagged recombinant angiocidin. All experiments were repeated three times to establish reproducibility. The experiments shown are representative of three experiments performed for each panel.

Fig. 6

The effect of angiocidin on fibronectin incorporation into ECM of HUVE cells. A- The effect of angiocidin and anti-tTgase antibody on biotin-cadaverine incorporation in HUVE-cells. The major band labeled with biotin-cadaverine was identified as fibronectin by Western-blot analysis. B- Immunohisto localization of fibronectin in untreated or HUVE cells treated with angiocidin or anti-tTgase antibody. Cells photographed at 100× magnification. C. Immunohisto localization of fibronectin in untreated or HUVE cells treated with angiocidin. Cells photographed at 400× magnification. All experiments were repeated three times to establish reproducibility. The experiments shown are representative of three experiments performed for each panel.

Fig. 7

Immunohistochemical analysis of isopeptide bond formation and his-tag angiocidin localization in control and angiocidin-treated tumor-bearing mice Tumor tissue of Lewis lung engrafted mice either treated with buffer or angiocidin was examined immunohistochemically for the presence of isopeptide bonds, His-tagged angiocidin and FN. Images were photographed at 100× magnification. All experiments were repeated three times to establish reproducibility. The experiments shown are representative of three experiments performed for each panel.