Polymeric Osteopontin Employs Integrin alpha 9 beta 1 as a Receptor and Attracts Neutrophils by Presenting a de Novo Binding Site

Nishimichi Norihisa

Higashikawa Fumiko

Kinoh Hiromi H

Tateishi Yoshiko

Matsuda Haruo

Yokosaki Yasuyuki

Journal of Bioligical Chemistry

Osteopontin (OPN) is a cytokine and ligand for multiple members of the integrin family. OPN undergoes the in vivo polymerization catalyzed by cross-linking enzyme transglutaminase 2, which consequently increases the bioactivity through enhanced interaction with integrins. The integrin alpha 9 beta 1, highly expressed on neutrophils, binds to the sequence SVVYGLR only after intact OPN is cleaved by thrombin. The SVVYGLR sequence appears to be cryptic in intact OPN because alpha 9 beta 1 does not recognize intact OPN. Because transglutaminase 2-catalyzed polymers change their physical and chemical properties, we hypothesized that the SVVYGLR site might also be exposed on polymeric OPN. As expected, alpha 9 beta 1 turned into a receptor for polymeric OPN, a result obtained by cell adhesion and migration assays with alpha 9-transfected cells and by detection of direct binding of recombinant soluble alpha 9 beta 1 with colorimetry and surface plasmon resonance analysis. Because the N-terminal fragment of thrombincleaved OPN, a ligand for alpha 9 beta 1, has been reported to attract neutrophils, we next examined migration of neutrophils to polymeric OPN using time-lapse microscopy. Polymeric OPN showed potent neutrophil chemotactic activity, which was clearly inhibited by anti-alpha 9 beta 1 antibody. Unexpectedly, mutagenesis studies showed that alpha 9 beta 1 bound to polymeric OPN independently of the SVVYGLR sequence, and further, SVVYGLR sequence of polymeric OPN was cryptic because SVVYGLR-specific antibody did not recognize polymeric OPN. These results demonstrate that polymerization of OPN generates a novel alpha 9 beta 1-binding site and that the interaction of this site with the alpha 9 beta 1 integrin is critical to the neutrophil chemotaxis induced by polymeric OPN.

Medical sciences [ 490 ]

eng

Amer Soc Biochemistry Molecular Biology Iec

[ISSN] 0021-9258

[DOI] 10.1074/jbc.M901515200

[NCID] AA00251083

[DOI] http://dx.doi.org/10.1074/jbc.M901515200