title: A crucial sequence for transglutaminase type 2 extracellular trafficking in renal tubular epithelial cells lies in its N-terminal beta-sandwich domain.
abstract: Transglutaminase type 2 (TG2) catalyzes the formation of an ε-(γ-glutamyl)-lysine isopeptide bond between adjacent peptides or proteins including those of the extracellular matrix (ECM). Elevated extracellular TG2 leads to accelerated ECM deposition and reduced clearance that underlie tissue scarring and fibrosis. The extracellular trafficking of TG2 is crucial to its role in ECM homeostasis; however, the mechanism by which TG2 escapes the cell is unknown as it has no signal leader peptide and therefore cannot be transported classically. Understanding TG2 transport may highlight novel mechanisms to interfere with the extracellular function of TG2 as isoform-specific TG2 inhibitors remain elusive. Mammalian expression vectors were constructed containing domain deletions of TG2. These were transfected into three kidney tubular epithelial cell lines, and TG2 export was assessed to identify critical domains. Point mutation was then used to highlight specific sequences within the domain required for TG2 export. The removal of β-sandwich domain prevented all TG2 export. Mutations of Asp(94) and Asp(97) within the N-terminal β-sandwich domain were identified as crucial for TG2 externalization. These form part of a previously identified fibronectin binding domain ((88)WTATVVDQQDCTLSLQLTT(106)). However, siRNA knockdown of fibronectin failed to affect TG2 export. The sequence (88)WTATVVDQQDCTLSLQLTT(106) within the β-sandwich domain of TG2 is critical to its export in tubular epithelial cell lines. The extracellular trafficking of TG2 is independent of fibronectin.
authors.affiliation: Academic Nephrology Unit (Sheffield Kidney Institute), University of Sheffield, Sheffield S10 2RZ, United Kingdom.
authors.forename: Che-Yi
authors.initials: CY
authors.lastname: Chou
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Streets
authors.forename: Philip F
authors.initials: PF
authors.lastname: Watson
authors.forename: Linghong
authors.initials: L
authors.lastname: Huang
authors.forename: Elisabetta A M
authors.initials: EA
authors.lastname: Verderio
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Johnson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017077:Culture Media, Conditioned; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D007684:Kidney Tubules / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D021381:Protein Transport; D034741:RNA, Small Interfering; D014162:Transfection; D011503:Transglutaminases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D017077:Culture Media, Conditioned; D017931:DNA Primers; D005353:Fibronectins; D034741:RNA, Small Interfering; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins