Molecular heterogeneity of follistatin, an activin-binding protein. Higher affinity of the carboxyl-terminal truncated forms for heparan sulfate proteoglycans on the ovarian granulosa cell

Follistatin (FS), an activin-binding protein, is a monomer derived from two polypeptide core sequences of 315 (FS-315) and 288 (FS-288) amino acids originated from alternatively spliced mRNA. To define the structural heterogeneity of native FS, we purified six molecular forms of FS from porcine ovaries. Protein chemical analysis revealed that the structural differences among the six isoforms were caused by truncation of the carboxyl-terminal region and/or the presence of carbohydrate chains, resulting in the formation of FS-315, FS-288, and FS composed of 303 amino acids (FS-303) in various forms of glycosylation on the two potential Asn-linked glycosylation sites. The majority of FS isolated from porcine ovaries was FS-303, which may have been derived from FS-315 by proteolytic cleavage of the 12 COOH-terminal amino acids. All six molecular species have almost the same activin binding activity (Kd = 540-680 pM). By contrast, the COOH-terminal truncated form, FS-288, showed much higher affinity for the rat granulosa cell surface than FS-303, whereas FS-315 had no affinity. FS-288 bound to heparan sulfate-Sepharose CL-4B, but FS-315 did not, suggesting that the truncated forms of FS bind to heparan sulfate proteoglycans on the cell. COS cells transfected with the FS-288 DNA expressed the FS-288 protein, which adhered to the cell surface, but cells transfected with the FS-315 DNA secreted the expressed protein into the medium, which did not bind to the cell surface. In rat anterior pituitary culture, FS-288 (ED50 = 2 ng/ml) was more potent in suppressing follicle-stimulating hormone release than FS-303 (ED50 = 10 ng/ml) and FS-315 (ED50 = 20 ng/ml). These results suggest that cell-associated FS traps activin more tightly in the matrix, thereby more effectively blocking the activity of activin on heparan sulfate proteoglycans of the cell surface and that cell-associated FS plays an important role in controlling the various actions of activin in a paracrine or autocrine manner.