THE HEPARIN-BINDING SITE OF FOLLISTATIN IS INVOLVED IN ITS INTERACTION WITH ACTIVIN

Whether the heparin-binding site of follistatin would interact with ac tivin has been examined. When a mixture of recombinant human follistat in-288 (rhFS-288) and -315 (rhFS-315) was applied to an activin-couple d affinity column, followed by stepwise elution of the column using 4M urea, 8M urea, 1M guanidine-HCl and 2M guanidine-HCl, rhFS-315 was el uted with 4M urea, while rhFS-288 was eluted with 2M guanidine-HCl. Th is finding implies that the carboxyl-terminal 27 amino acid extension of rhFS-315, which is not present in rhFS-288, affects the binding of follistatin with activin. Addition of heparin (50 mu g/ml) to the elut ion solvent caused rhFS-288 to elute with 4M urea, whereas rhFS-315 wa s not affected. These data suggest for the first time that these two s tructurally related follistatin molecules interact with activin by dif ferent modes of binding and, in the presence of heparin, the interacti on of rhFS-288 with activin is indistinguishable from that of rhFS-315 . Two analogs of rhFS-288 mutated at the heparin binding site were elu ted with 8M urea or 1M guanidine-KCl, distinct from the elution profil e of the intact rhFS-288. These results indicated that mutation at the heparin binding site alters the activin binding affinity. In addition , bioassay of the two mutants showed that they were less potent than t he rhFS-288. These findings suggest that the heparin binding site of f ollistatin also contributes to its binding for activin, and heparin ma y play an important role in the bioactivity of follistatin. (C) 1995 A cademic Press, Inc.