Thrombin Cleavage of Osteopontin Disrupts a Pro-chemotactic Sequence for Dendritic Cells, which is Compensated by the Release of its Pro-chemotactic C-Terminal Fragment [Glycobiology and Extracellular Matrices]

August 11th, 2014 by Shao, Z., Morser, J., Leung, L. L. K.

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, while OPNRAA-FL had substantially less activity, indicating the importance of RGD. 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. OPN-FLR168A had reduced activity and the double mutant OPNRAA-FLR168A had even lower activity, indicating that these functional domains accounted for most of OPN-FLs pro-chemotactic activity. OPN-CTF also possessed substantial pro-chemotactic activity which was fully expressed upon thrombin cleavage and its release from the intact protein, since 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 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.
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