Thiol-Disulfide Isomerization in Thrombospondin: Effects of Conformation and Protein Disulfide Isomerase

Abstract

Thiol-disulfide isomerization in thrombospondin may affect the function of this adhesive protein. Two assays were developed to analyze the determinants of thiol-disulfide exchange and to correlate this exchange with thrombospondin conformation. (1) A competitive immunoassay for the EDTA-conformation of thrombospondin was developed with monoclonal antibody D4.6. (2) The free thiol(s) in thrombospondin was labeled with [3H]N-ethylmaleimide (NEM) under various conditions (the presence or absence of calcium, temperature, and pH), and thrombin digests of the labeled protein were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Consistent with previous reports, thrombin digest fragments of 150, 120, 20, and 14 kD were observed, each with radioactivity under some condition, plus a 25-kD peptide that was not labeled. Sequence data for these fragments and comparisons of SDS-PAGE analyses under reducing and nonreducing conditions indicated that Cys974 was the free thiol. The appearance of thiol label in the 120-kD fragment was previously shown to be a consequence of thiol-disulfide exchange (J Biol Chem 265:17859,1990) and label was recovered in this peptide only under conditions (absence of calcium, 37°C and pH 8.4) that led to the appearance of the EDTA-conformation of thrombospondin. Additional evidence for the correlation of EDTA-conformation and thiol-disulfide exchange was the enhanced conversion of thrombospondin to its EDTA-conformation in the presence of protein disulfide isomerase and the inability of thrombospondin pretreated with NEM to attain the EDTA-conformation. Flow cytometry with antibody D4.6 revealed platelet-associated thrombospondin in the EDTA-conformation in the presence of calcium, suggesting that the EDTA-conformation is a physiological conformation that does not necessarily require EDTA.

The C-terminal CGHC motif of protein disulfide isomerase supports thrombosis

“…For studies of ATP secretion, purified α-thrombin from J.W. Fenton II (Center for Laboratories and Research, New York State Department of Health, Albany, New York, USA) was used (53).…”

Chlamydia attachment to mammalian cells requires protein disulfide isomerase

“…Many cellular molecules are rendered functionally active by the disulfide isomerase activity of PDI. Thrombospondin on the surface of platelets is activated by PDI so that it engages thrombin in the clotting cascade (Hotchkiss et al, 1996;Huang et al, 1997). PDI is also involved in controlling levels of mammalian cell membrane receptors, such as thyroid stimulating hormone receptor, by eliciting the shedding of extracellular domains (Couet et al, 1996).…”

The interaction of Thrombospondins with extracellular matrix proteins

“…A caveat here is that the TSP-4 was not purified in the presence of calcium. This may have resulted in an irreversible change in conformation like that seen in TSP-1 because both proteins have a free sulfhydryl group in their C-terminal globular domains (Huang et al 1997 ). Addition of purified TSP-4 to laminin significantly promotes neurite outgrowth as compared to substrates covered with laminin alone.…”