Transglutaminase 2-Catalyzed Intramolecular Cross-Linking of Osteopontin

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• 作者：Brian Christensen ; Elias D. Zachariae ; Carsten Scavenius ; S?ren Kl?verpris ; Claus Oxvig ; Steen V. Petersen ; Jan J. Enghild ; Esben S. S?rensen
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：January 19, 2016
• 年：2016
• 卷：55
• 期：2
• 页码：294-303
• 全文大小：618K
• ISSN：1520-4995

文摘

Osteopontin (OPN) is a multifunctional integrin-binding protein present in several tissues and body fluids. OPN is a substrate for the enzyme transglutaminase 2 (TG2), which catalyzes inter- and intramolecular cross-linking affecting the biological activity of the protein. Polymerization of OPN by intermolecular cross-linking has mostly been studied using relatively high TG2 concentrations, whereas the effect of lower concentrations of TG2 has remained unexplored. Here we show that TG2 at physiologically relevant concentrations predominantly catalyzes the formation of intramolecular cross-links in OPN. By site-directed mutagenesis and mass spectrometry, we demonstrate that Gln⁴² and Gln¹⁹³ serve as the primary amine acceptor sites for isopeptide bond formation. We find that Gln⁴² predominantly is linked to Lys⁴ and that Gln¹⁹³ participates in a cross-link with Lys¹⁵⁴, Lys¹⁵⁷, or Lys²³¹. The formation of specific isopeptide bonds was not dependent on OPN phosphorylation, and similar patterns of cross-linking were observed in human and mouse OPN. Furthermore, we find that OPN purified from human urine contains the Lys¹⁵⁴–Gln¹⁹³ isopeptide bond, indicating that intramolecular cross-linking of OPN occurs in vivo. Collectively, these data suggest that specific intramolecular cross-linking in the N- and C-terminal parts of OPN is most likely the dominant step in TG2-catalyzed modification of OPN.