Heparin and heparan sulfate (HS) are highly sulfated polysaccharides covalently bound to cell surface proteins, which directly interact with many extracellular proteins, including the transforming growth factor-β (TGFβ) family ligand antagonist, follistatin 288 (FS288). Follistatin neutralizes the TGFβ ligands, myostatin and activin A, by forming a nearly irreversible non-signaling complex by surrounding the ligand and preventing interaction with TGFβ receptors. The FS288-ligand complex has higher affinity than unbound FS288 for heparin/HS, which accelerates ligand internalization and lysosomal degradation; however, limited information is available for how FS288 interactions with heparin affect ligand binding. Using surface plasmon resonance (SPR) we show that preincubation of FS288 with heparin/HS significantly decreased the association kinetics for both myostatin and activin A with seemingly no effect on the dissociation rate. This observation is dependent on the heparin/HS chain length where small chain lengths less than degree of polymerization 10 (dp10) did not alter association rates but chain lengths >dp10 decreased association rates. In an attempt to understand the mechanism for this observation, we uncovered that heparin induced dimerization of follistatin. Consistent with our SPR results, we found that dimerization only occurs with heparin molecules >dp10. Small-angle X-ray scattering of the FS288 heparin complex supports that FS288 adopts a dimeric configuration that is similar to the FS288 dimer in the ligand-bound state. These results indicate that heparin mediates dimerization of FS288 in a chain-length-dependent manner that reduces the ligand association rate, but not the dissociation rate or antagonistic activity of FS288.

肝素和硫酸乙酰肝素 (HS) 是与细胞表面蛋白共价结合的高度硫酸化多糖，可直接与许多细胞外蛋白相互作用，包括转化生长因子-β (TGFβ) 家族配体拮抗剂 follistatin 288 (FS288)。Follistatin 通过包围配体并阻止与 TGFβ 受体相互作用形成几乎不可逆的非信号复合物，从而中和 TGFβ 配体、肌肉生长抑制素和激活素 A。FS288-配体复合物对肝素/HS的亲和力高于未结合的FS288，加速配体内化和溶酶体降解；然而，关于 FS288 与肝素的相互作用如何影响配体结合的信息有限。使用表面等离子共振 (SPR)，我们显示 FS288 与肝素/HS 的预孵育显着降低了肌肉生长抑制素和激活素 A 的结合动力学，似乎对解离速率没有影响。这一观察结果取决于肝素/HS 链长，其中小于聚合度 10 (dp10) 的小链长不会改变缔合率，但链长 >dp10 会降低缔合率。为了理解这一观察的机制，我们发现肝素诱导了卵泡抑素的二聚化。与我们的 SPR 结果一致，我们发现二聚化仅在肝素分子 >dp10 时发生。FS288 肝素复合物的小角 X 射线散射支持 FS288 采用与配体结合状态下的 FS288 二聚体相似的二聚体构型。