Abstract
The cytokine osteopontin (OPN) can be hydrolyzed by thrombin exposing a cryptic alpha(4)beta(1)/alpha(9)beta(1) integrin-binding motif (SVVYGLR), thereby acting as a potent cytokine for cells bearing these activated integrins. We show that purified milk OPN is a substrate for thrombin with a k(cat)/K(m) value of 1.14 x 10(5) m(-1) s(-1). Thrombin cleavage of OPN was inhibited by unsulfated hirugen (IC(50) = 1.2 +/- 0.2 microm), unfractionated heparin (IC(50) = 56.6 +/- 8.4 microg/ml) and low molecular weight (5 kDa) heparin (IC(50) = 31.0 +/- 7.9 microg/ml), indicating the involvement of both anion-binding exosite I (ABE-I) and anion-binding exosite II (ABE-II). Using a thrombin mutant library, we mapped residues important for recognition and cleavage of OPN within ABE-I and ABE-II. A peptide (OPN-(162-197)) was designed spanning the OPN thrombin cleavage site and a hirudin-like C-terminal tail domain. Thrombin cleaved OPN-(162-197) with a specificity constant of k(cat)/K(m) = 1.64 x 10(4) m(-1) s(-1). Representative ABE-I mutants (K65A, H66A, R68A, Y71A, and R73A) showed greatly impaired cleavage, whereas the ABE-II mutants were unaffected, suggesting that ABE-I interacts principally with the hirudin-like OPN domain C-terminal and contiguous to the thrombin cleavage site. Debye-Hückel slopes for milk OPN (-4.1 +/- 1.0) and OPN-(162-197) (-2.4 +/- 0.2) suggest that electrostatic interactions play an important role in thrombin recognition and cleavage of OPN. Thus, OPN is a bona fide substrate for thrombin, and generation of thrombin-cleaved OPN with enhanced pro-inflammatory properties provides another molecular link between coagulation and inflammation.
Highlights
The cytokine osteopontin (OPN) can be hydrolyzed by thrombin exposing a cryptic ␣41/␣91 integrin-binding motif (SVVYGLR), thereby acting as a potent cytokine for cells bearing these activated integrins
Thrombin cleavage of OPN was inhibited by unsulfated hirugen (IC50 ؍1.2 ؎ 0.2 M), unfractionated heparin (IC50 ؍ 56.6 ؎ 8.4 g/ml) and low molecular weight (5 kDa) heparin (IC50 ؍31.0 ؎ 7.9 g/ml), indicating the involvement of both anion-binding exosite I (ABE-I) and anion-binding exosite II (ABE-II)
Thrombin cleavage of human OPN (Arg168–Ser169) exposes a cryptic integrin binding motif, 162SVVYGLR168, that binds integrins ␣41, ␣47, and ␣91 (16 –19); this sequence is adjacent to the RGD domain
Summary
Materials—Human wild-type (WT) and alanine-substituted mutant thrombins were expressed, purified, and titrated with PPACK (D-Phe-Pro-Arg-chloromethylketone) as described previously [20]. Low molecular weight (LMW) heparin (5 kDa), porcine intestinal mucosal unfractionated heparin, PPACK, and unsulfated hirulog were from Sigma. The peptides SVVYGLR and OPN-(162–197) (SVVYGLR/SKSKKFQRPDIQYPDATDEDITSHMESEE) were synthesized, purified, and quantitated by the peptide synthesis facility at Stanford University School of Medicine
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