The molecular structure of corticotropin-induced secreted protein, a novel member of the thrombospondin family.
CISP (corticotropin-induced secreted protein) is a secreted protein recently purified in our laboratory from the conditioned medium of ACTH-treated bovine adrenocortical cells. Partial amino acid sequencing of CISP revealed homology with thrombospondins (TSPs), a family of adhesive proteins and in particular with TSP2. We report here the characterization of the molecular structure of CISP. Analysis of CISP by polyacrylamide gel electrophoresis in the absence or presence of SDS indicated an apparent molecular mass approximately equal to 600 kDa for the unreduced protein and an apparent molecular mass of 195 kDa after reduction by 2-mercaptoethanol. The sedimentation coefficient of CISP determined by ultracentrifugation on sucrose gradients was shifted from 9.7 S in the absence to 5.7 S in the presence of 2-mercaptoethanol. These data are consistent with a trimeric organization of the CISP molecule in which 195-kDa monomers would be linked together by disulfide bonds. The trimeric structure of CISP could be observed by rotary shadowing/electron microscopy, where CISP appeared to be composed of three equally electron-dense nodules and of a fourth nodule formed by the close association of three smaller fragments. The overall size of the molecule was 60 nm. We also observed that CISP is sulfated and glycosylated. Using glycosylation inhibitors, we could determine that CISP is synthesized as a 175-kDa core protein, is then matured into a 190-kDa high-mannose form and secreted as a 195-kDa mature protein. Inhibition of sulfation by chlorate did not prevent CISP secretion, whereas inhibition of glycosylation by tunicamycin blocked it. Taken together, these data indicate that the TSP2-related CISP molecule presents both structural and functional properties very similar to those of TSP1. CISP differs greatly, however, from TSP1 by the inducibility of its synthesis by cAMP.[1]References
- The molecular structure of corticotropin-induced secreted protein, a novel member of the thrombospondin family. Pellerin, S., Lafeuillade, B., Wade, R.H., Savona, C., Chambaz, E.M., Feige, J.J. J. Biol. Chem. (1993) [Pubmed]
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