Cell surface localization of tissue transglutaminase is dependent on a fibronectin-binding site in its N-terminal beta-sandwich domain.
Increasing evidence indicates that tissue transglutaminase (tTG) plays a role in the assembly and remodeling of extracellular matrices and promotes cell adhesion. Using an inducible system we have previously shown that tTG associates with the extracellular matrix deposited by stably transfected 3T3 fibroblasts overexpressing the enzyme. We now show by confocal microscopy that tTG colocalizes with pericellular fibronectin in these cells, and by immunogold electron microscopy that the two proteins are found in clusters at the cell surface. Expression vectors encoding the full-length tTG or a N-terminal truncated tTG lacking the proposed fibronectin-binding site (fused to the bacterial reporter enzyme beta-galactosidase) were generated to characterize the role of fibronectin in sequestration of tTG in the pericellular matrix. Enzyme-linked immunosorbent assay style procedures using extracts of transiently transfected COS-7 cells and immobilized fibronectin showed that the truncation abolished fibronectin binding. Similarly, the association of tTG with the pericellular matrix of cells in suspension or with the extracellular matrix deposited by cell monolayers was prevented by the truncation. These results demonstrate that tTG binds to the pericellular fibronectin coat of cells via its N-terminal beta-sandwich domain and that this interaction is crucial for cell surface association of tTG.
Gaudry
C A
CA
Department of Life Sciences, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, United Kingdom.
Verderio
E
E
Aeschlimann
D
D
Cox
A
A
Smith
C
C
Griffin
M
M
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology*; D004730:Endothelium, Vascular / Q000201:enzymology; D004790:Enzyme Induction; D005353:Fibronectins / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D007150:Immunohistochemistry; D051379:Mice; D016253:Microscopy, Immunoelectron; D011401:Promoter Regions, Genetic; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D011503:Transglutaminases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D005353:Fibronectins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D001616:beta-Galactosidase; D019204:GTP-Binding Proteins