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Plasma transglutaminase in hypertrophic chondrocytes: expression and cell-specific intracellular activation produce cell death and externalization.

Nurminskaya M, Magee C, Nurminsky D, Linsenmayer TF - J. Cell Biol. (1998)

Bottom Line: We now have isolated a full-length cDNA for this molecule, and confirmed that it is avian factor XIIIA.This externalization most likely is effected by cell death and subsequent lysis-effected by the transglutaminase itself.Non-hypertrophic cells transfected with the same construct do not show these degenerative changes.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.

ABSTRACT
We previously used subtractive hybridization to isolate cDNAs for genes upregulated in chick hypertrophic chondrocytes (Nurminskaya, M. , and T.F. Linsenmayer. 1996. Dev. Dyn. 206:260-271). Certain of these showed homology with the "A" subunit of human plasma transglutaminase (factor XIIIA), a member of a family of enzymes that cross-link a variety of intracellular and matrix molecules. We now have isolated a full-length cDNA for this molecule, and confirmed that it is avian factor XIIIA. Northern and enzymatic analyses confirm that the molecule is upregulated in hypertrophic chondrocytes (as much as eightfold). The enzymatic analyses also show that appreciable transglutaminase activity in the hypertrophic zone becomes externalized into the extracellular matrix. This externalization most likely is effected by cell death and subsequent lysis-effected by the transglutaminase itself. When hypertrophic chondrocytes are transfected with a cDNA construct encoding the zymogen of factor XIIIA, the cells convert the translated protein to a lower molecular weight form, and they initiate cell death, become permeable to macromolecules and eventually undergo lysis. Non-hypertrophic cells transfected with the same construct do not show these degenerative changes. These results suggest that hypertrophic chondrocytes have a novel, tissue-specific cascade of mechanisms that upregulate the synthesis of plasma transglutaminase and activate its zymogen. This produces autocatalytic cell death, externalization of the enzyme, and presumably cross-linking of components within the hypertrophic matrix. These changes may in turn regulate the removal and/or calcification of this hypertrophic matrix, which are its ultimate fates.

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(A) Amino acid alignment of the chicken (ChPTG) and  human (HFXIII) plasma transglutaminase. Asterisks indicate potential translation start sites in the chicken. Boxes mark the Arg-Gly residues of the potential activating sites at positions 46–47  and 87–88. Different sites are used by the chicken and human as  designated by the circles. (B) Immunoblot of extracts of hypertrophic chondrocytes transfected with the pFXIIIA-GFP construct and probed with an antibody against the GFP moiety (B).  At 24 h after transfection, only a single band was detected (of the  size predicted for the zymogen), whereas by 150 h another band  was also present, suggesting the conversion/activation of some of  the zymogen.
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Figure 6: (A) Amino acid alignment of the chicken (ChPTG) and human (HFXIII) plasma transglutaminase. Asterisks indicate potential translation start sites in the chicken. Boxes mark the Arg-Gly residues of the potential activating sites at positions 46–47 and 87–88. Different sites are used by the chicken and human as designated by the circles. (B) Immunoblot of extracts of hypertrophic chondrocytes transfected with the pFXIIIA-GFP construct and probed with an antibody against the GFP moiety (B). At 24 h after transfection, only a single band was detected (of the size predicted for the zymogen), whereas by 150 h another band was also present, suggesting the conversion/activation of some of the zymogen.

Mentions: Extracellular activation of the zymogen of human plasma transglutaminase—as occurs during blood clotting—involves thrombin-mediated cleavage of the A subunit at an Arg-Gly site at position 38 (Fig. 6 A, circle in human sequence). The transfections of the avian hypertrophic chondrocytes described above, however, raise the possibility that this cell type can activate the zymogen intracellularly. In the chicken plasma transglutaminase, the deduced amino acid sequence contains two Arg-Gly residues (at positions 46 and 87; see Fig. 6 A, boxes) that, by comparison to the human and other species, are potential sites for activation.


Plasma transglutaminase in hypertrophic chondrocytes: expression and cell-specific intracellular activation produce cell death and externalization.

Nurminskaya M, Magee C, Nurminsky D, Linsenmayer TF - J. Cell Biol. (1998)

(A) Amino acid alignment of the chicken (ChPTG) and  human (HFXIII) plasma transglutaminase. Asterisks indicate potential translation start sites in the chicken. Boxes mark the Arg-Gly residues of the potential activating sites at positions 46–47  and 87–88. Different sites are used by the chicken and human as  designated by the circles. (B) Immunoblot of extracts of hypertrophic chondrocytes transfected with the pFXIIIA-GFP construct and probed with an antibody against the GFP moiety (B).  At 24 h after transfection, only a single band was detected (of the  size predicted for the zymogen), whereas by 150 h another band  was also present, suggesting the conversion/activation of some of  the zymogen.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132883&req=5

Figure 6: (A) Amino acid alignment of the chicken (ChPTG) and human (HFXIII) plasma transglutaminase. Asterisks indicate potential translation start sites in the chicken. Boxes mark the Arg-Gly residues of the potential activating sites at positions 46–47 and 87–88. Different sites are used by the chicken and human as designated by the circles. (B) Immunoblot of extracts of hypertrophic chondrocytes transfected with the pFXIIIA-GFP construct and probed with an antibody against the GFP moiety (B). At 24 h after transfection, only a single band was detected (of the size predicted for the zymogen), whereas by 150 h another band was also present, suggesting the conversion/activation of some of the zymogen.
Mentions: Extracellular activation of the zymogen of human plasma transglutaminase—as occurs during blood clotting—involves thrombin-mediated cleavage of the A subunit at an Arg-Gly site at position 38 (Fig. 6 A, circle in human sequence). The transfections of the avian hypertrophic chondrocytes described above, however, raise the possibility that this cell type can activate the zymogen intracellularly. In the chicken plasma transglutaminase, the deduced amino acid sequence contains two Arg-Gly residues (at positions 46 and 87; see Fig. 6 A, boxes) that, by comparison to the human and other species, are potential sites for activation.

Bottom Line: We now have isolated a full-length cDNA for this molecule, and confirmed that it is avian factor XIIIA.This externalization most likely is effected by cell death and subsequent lysis-effected by the transglutaminase itself.Non-hypertrophic cells transfected with the same construct do not show these degenerative changes.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.

ABSTRACT
We previously used subtractive hybridization to isolate cDNAs for genes upregulated in chick hypertrophic chondrocytes (Nurminskaya, M. , and T.F. Linsenmayer. 1996. Dev. Dyn. 206:260-271). Certain of these showed homology with the "A" subunit of human plasma transglutaminase (factor XIIIA), a member of a family of enzymes that cross-link a variety of intracellular and matrix molecules. We now have isolated a full-length cDNA for this molecule, and confirmed that it is avian factor XIIIA. Northern and enzymatic analyses confirm that the molecule is upregulated in hypertrophic chondrocytes (as much as eightfold). The enzymatic analyses also show that appreciable transglutaminase activity in the hypertrophic zone becomes externalized into the extracellular matrix. This externalization most likely is effected by cell death and subsequent lysis-effected by the transglutaminase itself. When hypertrophic chondrocytes are transfected with a cDNA construct encoding the zymogen of factor XIIIA, the cells convert the translated protein to a lower molecular weight form, and they initiate cell death, become permeable to macromolecules and eventually undergo lysis. Non-hypertrophic cells transfected with the same construct do not show these degenerative changes. These results suggest that hypertrophic chondrocytes have a novel, tissue-specific cascade of mechanisms that upregulate the synthesis of plasma transglutaminase and activate its zymogen. This produces autocatalytic cell death, externalization of the enzyme, and presumably cross-linking of components within the hypertrophic matrix. These changes may in turn regulate the removal and/or calcification of this hypertrophic matrix, which are its ultimate fates.

Show MeSH
Related in: MedlinePlus