title: 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.
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.
authors.affiliation: Division of Hematology, Stanford University School of Medicine, Stanford, California 94305 and; Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304.
authors.forename: Zhifei
authors.initials: Z
authors.lastname: Shao
authors.affiliation: Division of Hematology, Stanford University School of Medicine, Stanford, California 94305 and; Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304.
authors.forename: John
authors.initials: J
authors.lastname: Morser
authors.affiliation: Division of Hematology, Stanford University School of Medicine, Stanford, California 94305 and; Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304. Electronic address: lawrence.leung@stanford.edu.
authors.forename: Lawrence L K
authors.initials: LLK
authors.lastname: Leung
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D002465:Cell Movement / Q000502:physiology*; D003713:Dendritic Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D018945:Monocyte Chemoattractant Proteins / Q000235:genetics / Q000378:metabolism*; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis; D013917:Thrombin / Q000235:genetics / Q000378:metabolism*
chemical_list: C497483:CD44 protein, human; C491224:Ccl12 protein, mouse; C476399:Cd44 protein, mouse; D018960:Hyaluronan Receptors; D018945:Monocyte Chemoattractant Proteins; C506896:Spp1 protein, mouse; D053495:Osteopontin; D013917:Thrombin
keywords: Chemokine; Dendritic Cell; Migration; Osteopontin; Thrombin
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