THE MOLECULAR-STRUCTURE OF CORTICOTROPIN-INDUCED SECRETED PROTEIN, A NOVEL MEMBER OF THE THROMBOSPONDIN FAMILY

CISP (corticotropin-induced secreted protein) is a secreted Protein re cently 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 adhesi ve proteins and in particular with TSP2. We report here the characteri zation of the molecular structure of CISP. Analysis of CISP by polyacr ylamide gel electrophoresis in the absence or presence of SDS indicate d an apparent molecular mass congruent-to 600 kDa for the unreduced pr otein and an apparent molecular mass of 195 kDa after reduction by 2-m ercaptoethanol. The sedimentation coefficient of CISP determined by ul tracentrifugation on sucrose gradients was shifted from 9.7 S in the a bsence to 5.7 S in the presence of 2-mercaptoethanol. These data are c onsistent with a trimeric organization of the CISP molecule in which 1 95-kDa monomers would be linked together by disulfide bonds. The trime ric structure of CISP could be observed by rotary shadowing/electron m icroscopy, where CISP appeared to be composed of three equally electro n-dense nodules and of a fourth nodule formed by the close association of three smaller fragments. The overall size of the molecule was 60 n m. We also observed that CISP is sulfated and glycosylated. Using glyc osylation inhibitors, we could determine that CISP is synthesized as a 175-kDa core protein, is then matured into a 190-kDa high-mannose for m and secreted as a 195-kDa mature protein. Inhibition of sulfation by chlorate did not prevent CISP secretion, whereas inhibition of glycos ylation by tunicamycin blocked it. Taken together, these data indicate that the TSP2-related CISP molecule presents both structural and func tional properties very similar to those of TSP1. CISP differs greatly, however, from TSP1 by the inducibility of its synthesis by cAMP.