Characterization of Heparin-binding Site of Tissue Transglutaminase: ITS IMPORTANCE IN CELL SURFACE TARGETING, MATRIX DEPOSITION, AND CELL SIGNALING

Zhuo Wang,Russell J Collighan,Kamila Pytel,Daniel L Rathbone,Xiaoling Li,Martin Griffin

doi:10.1074/jbc.m111.294819

Abstract

Tissue transglutaminase (TG2) is a multifunctional Ca(2+)-activated protein cross-linking 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 nontransamidating mechanism via its association with fibronectin, heparan sulfates (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 modeling 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 for the RGD-induced loss of cell adhesion on fibronectin 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.

Highlights

TG2 is a multifunctional matrix protein and cross-linking enzyme
By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the heparan sulfate (HS)-binding site within TG2, we demonstrate how this mimicking peptide can in isolation compensate for the RGD-induced loss of cell adhesion on fibronectin via binding to syndecan-4, leading to activation of PKC␣, pFAK-397, and extracellular signal-regulated kinase1/2 (ERK1/2) and the subsequent formation of focal adhesions and actin cytoskeleton organization
Identification of Putative Heparin-binding Motifs in TG2— HS-binding motifs may be composed of basic amino acid-containing sequences, such as XBBXBX or XBBBXXBX, where B is a basic amino acid whose side chain is exposed on the protein surface and X is a neutral or hydrophobic amino acid, whose side chain is directed toward the protein interior [23]

Summary

Introduction

Results: Identification and mutation of its heparan sulfate (HS)-binding site blocks matrix deposition of TG2 as do inhibitors of syndecan shedding. Significance: Blocking heparan sulfate binding provides an avenue for regulating the pathological roles of the enzyme. Tissue transglutaminase (TG2) is a multifunctional Ca2؉-activated protein cross-linking 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 facilitate cell adhesion important in wound healing through a nontransamidating mechanism via its association with fibronectin, heparan sulfates (HS), and integrins. Regulating the mechanism how TG2 is translocated into the ECM provides a strategy for modulating these physiological and pathological functions of the enzyme. We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed

Objectives

Results

Conclusion