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Substrate‐dependent localization of transglutaminase 2
Author(s) -
Akker Jeroen,
Afink Gijs B,
Weert Angela,
Vos Judith,
Bakker Erik NTP,
vanBavel Ed
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.822.1
Subject(s) - hek 293 cells , fibronectin , transfection , tissue transglutaminase , microbiology and biotechnology , green fluorescent protein , cytosol , cell culture , western blot , cell adhesion , cell , extracellular matrix , chemistry , biology , biochemistry , gene , enzyme , genetics
Transglutaminase 2 (TG2) is known to be important for both cross‐linking of matrix proteins as well as cell adhesion, cytoskeletal organization and signaling. However, its cellular distribution and mechanisms of secretion and activation during matrix remodeling remain unclear. We tagged TG2 at the C‐terminal with eGFP. TG2/eGFP was transfected into HEK/293T cells using Effectene. Western blot and in vitro activity assay showed a >100‐fold increase in active TG2. No difference was observed in TG2 activity between TG2/eGFP and its non‐fluorescent control. TG2 distribution in HEK cells strongly depended on the underlying substrate. When seeded on collagen type I, TG2 appeared mainly as intense intracellular and membrane‐bound spots, as well as in microparticles at distances up to 100 micron from the cell. However, on a fibronectin coating, TG2 was rather homogenously distributed throughout the cytosol. In HEK cells plated on plastic, TG2 localized strongly along the cell membrane. Here, transfection induced a large increase in fibronectin staining, which colocalized with TG2 fluorescence. TG2 was almost absent in the nuclei of living cells. In conclusion, we developed a construct of green fluorescing TG2. HEK cells expressing TG2/eGFP showed distinct localization of TG2, depending on its ECM. Only TG2‐transfected cells were able to produce a membrane‐bound fibronectin matrix. Supported by NHF grant 2005.B080