Thrombospondin 1 (TSP1) is a 450 kDa homotrimeric multidomain glycoprotein with fundamental roles in many cell-cell and cell-matrix interactions. These varied, and sometimes conflicting, functions are mediated by specific domains in TSP1. One region with diverse biological roles is the Ca2+ binding loops (or type 3 repeats). The biological activity of this region is determined through a complex assembly of disulfide bonds linking structure and function. Disulfide interchange in a protein is usually very specific and quite slow, unless catalysed. I have found that protein disulfide isomerase (PDI) is expressed on the surface of platelets and endothelial cells in a reduced active conformation. The presence of enzymatically active PDI on the surface of TSP1-secreting cells suggests PDI is well positioned to catalyse disulfide interchange in, and regulate the structure/function relationships of, TSP1. PDI was observed to form disulfide-linked complexes with TSP1. Moreover, incubation of platelet or fibroblast TSP1 with PDI enhanced binding of an isomer-specific anti-TSP1 antibody whose epitope is in the Ca2+ binding loops. These findings suggest that PDI may mediate disulfide bond rearrangement in both the soluble and extracellular matrix-bound forms of TSP1. TSP1 is a tight-binding competitive inhibitor of neutrophil cathepsin G; however, incubation with PDI increased the Ki for the interaction ???10-14-fold. TSP1 bound platelet-derived growth factor (PDGF) tightly in the region of the Ca2+ binding loops and supported binding of PDGF to its receptor. PDI-mediated disulfide interchange in TSP1 ablated PDGF binding, indicating that PDI-catalysed disulfide interchange in TSP1 may modulate PDGF-TSP1 complex formation and the biological activity of PDGF. Finally, PDI-catalysed isomerization of TSP1 potently affected its cell adhesive properties. Treatment of TSP1 with PDI enhanced adhesion and spreading of endothelial cells through the ??v??3 integrin receptor to TSP1, by exposure of a cryptic RGD sequence. Thus, endothelial cell surface PDI may be a physiological regulator of RGD-dependent binding to TSP1. These data suggest that cell-surface PDI may regulate the disulfide-bonded structure and certain biological functions of TSP1. In conclusion, I propose a novel mechanism for the post-translational regulation of TSP1 structure/function, which in turn may regulate certain aspects of TSP1 in vascular biology.
| Identifer | oai:union.ndltd.org:ADTP/273578 |
| Date | January 2009 |
| Creators | Hotchkiss, Kylie A, Medical Sciences, Faculty of Medicine, UNSW |
| Publisher | Awarded by:University of New South Wales. Medical Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Hotchkiss Kylie A., http://unsworks.unsw.edu.au/copyright |
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