Thacker Bryan E, Seamen Emylie, Lawrence Roger, Parker Matthew W, Xu Yongmei, Liu Jian, Vander Kooi Craig W, Esko Jeffrey D
Center for Structural Biology, Department of Molecular and Cellular Biochemistry, University of Kentucky , Lexington, Kentucky 40536, United States.
Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina , Chapel Hill, North Carolina 27599, United States.
ACS Chem Biol. 2016 Apr 15;11(4):971-80. doi: 10.1021/acschembio.5b00897. Epub 2016 Jan 14.
Binding of proteins to heparan sulfate is driven predominantly by electrostatic interactions between positively charged amino acid residues in the protein and negatively charged sulfate groups located at various positions along the polysaccharide chain. Although many heparin/heparan-sulfate-binding proteins have been described, few exhibit preferential binding for heparan sulfates containing relatively rare 3-O-sulfated glucosamine residues. To expand the "3-O-sulfate proteome," affinity matrices were created from Chinese hamster ovary (CHO) cell heparan sulfate engineered in vitro with and without 3-O-sulfate groups. Fractionation of different animal sera yielded several proteins that bound specifically to columns containing 3-O-sulfated heparan sulfate modified by two members of the heparan sulfate 3-O-sulfotransferase superfamily, Hs3st1 and Hs3st2. Neuropilin-1 was analyzed in detail because it has been implicated in angiogenesis and axon guidance. We show that 3-O-sulfation enhanced the binding of neuropilin-1 to heparan sulfate immobilized on plastic plates and to heparan sulfate present on cultured cells. Chemoenzymatically synthesized 3-O-sulfated heparan sulfate dodecamers protected neuropilin-1 from thermal denaturation and inhibited neuropilin-1-dependent, semaphorin-3a-induced growth cone collapse of neurons derived from murine dorsal root ganglia. The effect of 3-O-sulfation was cell autonomous and specific to Hs3st2 based on collapse assays of neurons derived from Hs3st1- and Hs3st2-deficient mice. Finally, 3-O-sulfated heparan sulfate enhanced the inhibition of endothelial cell sprouting by exogenous heparan sulfate. These findings demonstrate a reliable method to identify members of the 3-O-sulfate proteome and that 3-O-sulfation of heparan sulfate can modulate axonal growth cone collapse and endothelial cell sprouting.
蛋白质与硫酸乙酰肝素的结合主要由蛋白质中带正电荷的氨基酸残基与多糖链上不同位置带负电荷的硫酸基团之间的静电相互作用驱动。尽管已经描述了许多肝素/硫酸乙酰肝素结合蛋白，但很少有蛋白对含有相对罕见的3-O-硫酸化葡糖胺残基的硫酸乙酰肝素表现出优先结合。为了扩展“3-O-硫酸化蛋白质组”，利用体外工程改造的含有和不含有3-O-硫酸基团的中国仓鼠卵巢（CHO）细胞硫酸乙酰肝素制备了亲和基质。对不同动物血清进行分级分离，得到了几种与含有由硫酸乙酰肝素3-O-磺基转移酶超家族的两个成员Hs3st1和Hs3st2修饰的3-O-硫酸化硫酸乙酰肝素的柱子特异性结合的蛋白质。对神经纤毛蛋白-1进行了详细分析，因为它与血管生成和轴突导向有关。我们发现，3-O-硫酸化增强了神经纤毛蛋白-1与固定在塑料板上的硫酸乙酰肝素以及培养细胞上存在的硫酸乙酰肝素的结合。化学酶法合成的3-O-硫酸化硫酸乙酰肝素十二聚体保护神经纤毛蛋白-1免受热变性，并抑制神经纤毛蛋白-1依赖的、信号素-3a诱导的源自小鼠背根神经节的神经元生长锥塌陷。基于对源自Hs3st1和Hs3st2缺陷小鼠的神经元的塌陷分析，3-O-硫酸化的作用是细胞自主的且对Hs3st2具有特异性。最后，3-O-硫酸化硫酸乙酰肝素增强了外源性硫酸乙酰肝素对内皮细胞芽生的抑制作用。这些发现证明了一种鉴定3-O-硫酸化蛋白质组成员的可靠方法，并表明硫酸乙酰肝素的3-O-硫酸化可以调节轴突生长锥塌陷和内皮细胞芽生。
