Metastasis-associated S100A4 is a specific amine donor and an activity-independent binding partner of transglutaminase-2

Abstract

Transglutaminase-2 (TG2) is best known as a Ca 2+ -dependent cross-linking enzyme; however, some of its extracellular matrix-related functions are independent from its catalytic activity and include matrix remodeling, adhesion and migration. S100A4 belongs to the Ca 2+ -binding EF-hand S100 protein family and acts both intra-and extracellularly through binding to various partners. It regulates cell migration and its overexpression is strongly associated with metastasis and poor survival in various cancers. TG2 has recently been suggested to mediate S100A4-dependent tumor cell migration. Here we provide evidence that S100A4 is an interacting partner and also specific amine donor of TG2. TG2 incorporates a glutamine donor peptide to Lys100 in the C-terminal random coil region of S100A4. Importantly, the enzyme activity is not necessary for the interaction: S100A4 also binds to TG2 in the presence of a specific inhibitor that keeps the enzyme in an open conformation, or to an enzymatically inactive mutant. We also found that S100A4 considerably enhances TG2-mediated adhesion of A431 epithelial carcinoma cells to the extracellular matrix. This role is independent of enzyme activity and requires the open conformation of TG2. We propose that S100A4 stabilizes the open conformation of TG2, which binds to its cell surface receptor in this state and increases cell adhesion.Summary: S100A4 and transglutaminase-2 have a role in metastasis. S100A4 is an interaction partner and specific amine substrate of transglutaminase-2, promoting its open conformation and leading to enhanced cell adhesion. Studying their interaction could contribute to the better understanding of metastasis.Running title: S100A4: substrate and binding partner of transglutaminase-2

Real-time kinetic method to monitor isopeptidase activity of transglutaminase 2 on protein substrate

“…S1B). In the transamidase reaction the K m values for S100A4(GST) and BSA were comparable with earlier observations [15,18], while V max was approximately 10 times higher in case of S100A4(GST) as amine donor (Fig. S1C) suggesting that S100A4(GST) could be a very potent Lys donor to produce an isopeptidase protein substrate with a single isopeptide bond.…”

“…The FLpepT26 peptide was obtained as published in [18]. ZDON was sold by Zedira (Darmstadt, Germany).…”

“…By enzymatic crosslinking of the fluorescently labelled FLpepT26 dodecapeptide and S100A4, a recently characterized specific amine donor of TG2 [18], the purified product was subsequently used as a substrate to demonstrate the cleavage of the isopeptide bond and follow this activity by measuring fluorescence polarization in real time.…”

“…In previous publications fluorescein labelled FLpepT26 peptide was an effective TG2 specific amine acceptor substrate [15] that could be incorporated into the amine donor BSA and recently into S100A4 monitored by fluorescence polarisation assay [18]. Both FLpepT26-BSA and FLpepT26-S100A4 as N ε -(γ-glutamyl)lysine cross-linked products were considered as substrates for isopeptidase activity measurements.…”

“…However, BSA contains several surface exposed Lys residues compared to S100A4 which has only one TG2 reactive Lys residue (Lys100), and unlike BSA, S100A4 does not contain transglutaminase reactive glutamine residues [18] making S100A4 an ideal candidate to serve as a substrate in an isopeptidase reaction. To compensate the size difference between S100A4 (11.5 kDa) and BSA and to get higher anisotropy change and better fluorescence polarization signal in the designed assay, S100A4 was expressed with GST-tag.…”