Abstract
Thrombin cleavage alters the function of osteopontin (OPN) by exposing an integrin binding site and releasing a chemotactic C-terminal fragment. Here, we examined thrombin cleavage of OPN in the context of dendritic cell (DC) migration to define its functional domains. Full-length OPN (OPN-FL), thrombin-cleaved N-terminal fragment (OPN-R), thrombin- and carboxypeptidase B2-double-cleaved N-terminal fragment (OPN-L), and C-terminal fragment (OPN-CTF) did not have intrinsic chemotactic activity, but all potentiated CCL21-induced DC migration. OPN-FL possessed the highest potency, whereas OPNRAA-FL had substantially less activity, indicating the importance of RGD. We identified a conserved (168)RSKSKKFRR(176) sequence on OPN-FL that spans the thrombin cleavage site, and it demonstrated potent pro-chemotactic effects on CCL21-induced DC migration. OPN-FLR168A had reduced activity, and the double mutant OPNRAA-FLR168A had even lower activity, indicating that these functional domains accounted for most of the pro-chemotactic activity of OPN-FL. OPN-CTF also possessed substantial pro-chemotactic activity, which was fully expressed upon thrombin cleavage and its release from the intact protein, because OPN-CTF was substantially more active than OPNRAA-FLR168A containing the OPN-CTF sequence within the intact protein. OPN-R and OPN-L possessed similar potency, indicating that the newly exposed C-terminal SVVYGLR sequence in OPN-R was not involved in the pro-chemotactic effect. OPN-FL and OPN-CTF did not directly bind to the CD44 standard form or CD44v6. In conclusion, thrombin cleavage of OPN disrupts a pro-chemotactic sequence in intact OPN, and its loss of pro-chemotactic activity is compensated by the release of OPN-CTF, which assumes a new conformation and possesses substantial activity in enhancing chemokine-induced migration of DCs.
Highlights
Osteopontin is a chemotactic protein for dendritic cells without a clear definition of its functional domains
Neither OPN nor Its Fragments Induce Migration of Mature dendritic cell (DC)—Because OPN had previously been shown to induce mouse DC [3] and macrophage [19] chemotaxis, we first tested the potency of OPN in inducing human DC migration in comparison with the lymphatic chemokine CCL21 in a transmigration assay
We demonstrated the role of OPN as an enhancer of CC-chemokine-induced DC migration while not being a chemoattractant by itself
Summary
Osteopontin is a chemotactic protein for dendritic cells without a clear definition of its functional domains. Thrombin cleavage alters the function of osteopontin (OPN) by exposing an integrin binding site and releasing a chemotactic C-terminal fragment. We identified a conserved 168RSKSKKFRR176 sequence on OPN-FL that spans the thrombin cleavage site, and it demonstrated potent pro-chemotactic effects on CCL21-induced DC migration. Upon antigen uptake and activation, DCs migrate to lymph nodes, a process primarily driven by engagement of chemokines CCL19 and CCL21 with their common receptor CCR7 [21] Because both DCs [6, 22] and T cells [23] are sources of OPN that may play a role in DC migration, in this study, we investigated the impact of thrombin cleavage of OPN on DC migration to identify the functional domains of OPN. Thrombin cleavage disrupts its integrity, leading to a significant reduction in its pro-chemotactic function, but this is compensated by the released OPN-CTF that may assume a new conformation and possesses substantial pro-chemotactic activity
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