Polymeric osteopontin employs integrin alpha9beta1 as a receptor and attracts neutrophils by presenting a de novo binding site.
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 alpha9beta1, 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 alpha9beta1 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, alpha9beta1 turned into a receptor for polymeric OPN, a result obtained by cell adhesion and migration assays with alpha9-transfected cells and by detection of direct binding of recombinant soluble alpha9beta1 with colorimetry and surface plasmon resonance analysis. Because the N-terminal fragment of thrombin-cleaved OPN, a ligand for alpha9beta1, 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-alpha9beta1 antibody. Unexpectedly, mutagenesis studies showed that alpha9beta1 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 alpha9beta1-binding site and that the interaction of this site with the alpha9beta1 integrin is critical to the neutrophil chemotaxis induced by polymeric OPN.
Cell-Matrix Frontier Laboratory, Biomedical Research Unit, Hiroshima University, 1-2-3 Kasumi, Minamiku, Hiroshima 734-8551, Japan.
Norihisa
N
Nishimichi
Fumiko
F
Higashikawa
Hiromi H
HH
Kinoh
Yoshiko
Y
Tateishi
Haruo
H
Matsuda
Yasuyuki
Y
Yokosaki
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001665:Binding Sites; D001704:Biopolymers / Q000378:metabolism*; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002465:Cell Movement; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000166:cytology* / Q000378:metabolism; D053495:Osteopontin / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D001704:Biopolymers; D016023:Integrins; D011994:Recombinant Proteins; C113256:integrin alpha 9 beta 1; D053495:Osteopontin