CATALYSIS OF DISULFIDE ISOMERIZATION IN THROMBOSPONDIN-1 BY PROTEIN DISULFIDE-ISOMERASE

Thrombospondin 1 is a multidomain glycoprotein from platelets and most cells that participates in diverse biological processes. The structur e and some functional properties of thrombospondin 1 are regulated by disulfide interchange in the Ca2+-binding repeats and C-globular domai n. The recent identification of the enzyme, protein disulfide isomeras e, on the platelet surface suggested that protein disulfide isomerase may catalyze disulfide isomerization in platelet thrombospondin 1. Pro tein disulfide isomerase was found to form disulfide-linked complexes with thrombospondin 1, which is consistent with protein disulfide isom erase-mediated rearrangement of disulfide bonds in thrombospondin 1. T o quantitate disulfide interchange in thrombospondin 1, perturbation o f the enzyme inhibitory properties of platelet thrombospondin I were m easured, specifically changes in the apparent dissociation constant fo r inhibition of neutrophil cathepsin G by thrombospondin 1. The inhibi tion constant increased greater than or equal to 10-14-fold following incubation of either Ca2+-replete or Ca2+-depleted thrombospondin 1 wi th protein disulfide isomerase and reduced glutathione, The rate of pr otein disulfide isomerase-catalyzed disulfide interchange in thrombosp ondin 1 increased linearly with protein disulfide isomerase concentrat ion and the K-m for reduced glutathione was 0.4 +/- 0.2 mM, Disulfide isomerization in both platelet and fibroblast thrombospondin 1 was pro bed by measuring perturbation in epitopes for two anti-thrombospondin 1 monoclonal antibodies. Antibody D4.6 binds to the C-terminal Ca2+-bi nding domains which are involved in disulfide interchange, whereas ant ibody HB8432 binds toward the N-terminus of the thrombospondin 1 subun it. In accordance with the location of these epitopes, incubation of p latelet thrombospondin 1 or fibroblast thrombospondin 1 with protein d isulfide isomerase and reduced glutathione resulted in 2-fold enhancem ent of binding of D4.6, whereas binding of HB8432 did not significantl y change. In summary, protein disulfide isomerase catalyzes disulfide interchange in thrombospondin 1 which alters binding of neutrophil cat hepsin G and antibody D4.6 to thrombospondin 1.