The heparin binding site of follistatin is involved in its interaction with activin.
Whether the heparin-binding site of follistatin would interact with activin has been examined. When a mixture of recombinant human follistatin-288 (rhFS-288) and -315 (rhFS-315) was applied to an activin-coupled affinity column, followed by stepwise elution of the column using 4M urea, 8M urea, 1M guanidine-HCl and 2M guanidine-HCl, rhFS-315 was eluted with 4M urea, while rhFS-288 was eluted with 2M guanidine-HCl. This finding implies that the carboxylterminal 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 micrograms/ml) to the elution solvent caused rhFS-288 to elute with 4M urea, whereas rhFS-315 was not affected. These data suggest for the first time that these two structurally related follistatin molecules interact with activin by different modes of binding and, in the presence of heparin, the interaction of rhFS-288 with activin is indistinguishable from that of rhFS-315. Two analogs of rhFS-288 mutated at the heparin binding site were eluted with 8M urea or 1M guanidine-HCl, distinct from the elution profile 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 the rhFS-288. These findings suggest that the heparin binding site of follistatin also contributes to its binding for activin, and heparin may play an important role in the bioactivity of follistatin.
Sumitomo
S
S
Department of Molecular Endocrinology, Whittier Institute for Diabetes and Endocrinology, La Jolla, California 92037.
Inouye
S
S
Liu
X J
XJ
Ling
N
N
Shimasaki
S
S
D028341:Activins; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001681:Biological Assay; D015153:Blotting, Western; D016466:CHO Cells; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D006224:Cricetinae; D017931:DNA Primers; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D038681:Follistatin; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D006133:Growth Substances / Q000378:metabolism; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007265:Inhibins / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection
D017931:DNA Primers; D038681:Follistatin; D006023:Glycoproteins; D006133:Growth Substances; D011994:Recombinant Proteins; D028341:Activins; D007265:Inhibins; D005640:Follicle Stimulating Hormone; D006493:Heparin