Characterization of the heparin binding site of tissue transglutaminase: its importance in the enzyme’s cell surface targeting, matrix deposition and cell signalling

Tissue transglutaminase (TG2) is a multifunctional Ca 2+ activated protein crosslinking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a non-transamidating mechanism via its association with fibronectin (FN), heparan sulphates (HS) and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modelling and mutagenesis we have identified the HS binding site of TG2 202 KFLKNAGRDCSRRSSPVYVGR 222 . We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate the RGD-induced loss of cell adhesion on FN via binding to syndecan-4, leading to activation of PKCα, pFAK-397 and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed. Abbreviations: TG2, Tissue transglutaminase; ECM, Extracellular matrix; FN, Fibronectin; HSPG, Heparan sulphate proteoglycans; HS, Heparan sulphate; MMPs, Matrix metalloproteinases; ERK1/2, Extracellular-signal regulated kinase1/2; HOB, Human osteoblasts; MEF, mouse embryonic fibroblast; gplTG, Guinea pig liver TG; tetracycline-inducible, tet-inducible; NS, Non-silencing; PMA, α-phorbol-12, 13didecanoate; PKCα, Protein kinase C α; FAK, focal adhesion kinase; CHO-K1, Chinese hamster ovary-K1; DMEM, Dulbecco’s modified Eagle’s medium; FBS, foetal bovine serum; α-Tbl,αTubulin.