Xu Ding, Song Danyin, Pedersen Lars C, Liu Jian
Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
J Biol Chem. 2007 Mar 16;282(11):8356-67. doi: 10.1074/jbc.M608062200. Epub 2007 Jan 15.
Heparan sulfate (HS) and chondroitin sulfate (CS) are highly sulfated polysaccharides with a wide range of biological functions. Heparan sulfate 2-O-sulfotransferase (HS-2OST) transfers the sulfo group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the 2-OH position of the hexauronic acid that is adjacent to N-sulfated glucosamine, whereas chondroitin sulfate 2-O-sulfotransferase (CS-2OST) transfers the sulfo group to the hexauronic acid that is adjacent to N-acetylated galactosamine. Here we report a systematic mutagenesis study of HS-2OST and CS-2OST based on their structural homology to estrogen sulfotransferase and HS 3-O-sulfotransferase isoform 3 (3-OST3), for which crystal structures exist. We have identified six residues possibly involved in binding to PAPS. HS-2OST carrying mutations of these residues lacks sulfotransferase activity and the ability to bind 3'-phosphoadenosine 5'-phosphate, a PAPS analogue, as determined by isothermal titration calorimetry. Similar residues involved in binding to PAPS were also identified in CS-2OST. Additional residues that participate in carbohydrate substrate binding were also identified in both enzymes. Mutations at these residues led to the loss of sulfotransferase activity but maintained the ability to bind to phosphoadenosine 5'-phosphate. The catalytic function of HS-2OST appears to involve two histidine residues (His140 and His142), whereas only one histidine (His168) of CS 2-OST is likely to be critical. This unique feature of HS 2-OST catalytic residues directed us to characterize the Drosophila heparan sulfate 2-O-sulfotransferase. The results from this study provide insight into the differences and similarities various residues play in the biological roles of the HS-2OST and CS-2OST enzymes.
硫酸乙酰肝素（HS）和硫酸软骨素（CS）是具有广泛生物学功能的高度硫酸化多糖。硫酸乙酰肝素2-O-磺基转移酶（HS-2OST）将磺基从3'-磷酸腺苷5'-磷酸硫酸酯（PAPS）转移至与N-硫酸化葡糖胺相邻的己糖醛酸的2-OH位置，而硫酸软骨素2-O-磺基转移酶（CS-2OST）则将磺基转移至与N-乙酰化半乳糖胺相邻的己糖醛酸。在此，我们基于HS-2OST和CS-2OST与雌激素磺基转移酶以及硫酸乙酰肝素3-O-磺基转移酶同工型3（3-OST3）的结构同源性报告了一项系统的诱变研究，已知这两种酶存在晶体结构。我们已鉴定出六个可能参与与PAPS结合的残基。携带这些残基突变的HS-2OST缺乏磺基转移酶活性以及结合3'-磷酸腺苷5'-磷酸（一种PAPS类似物）的能力，这通过等温滴定量热法得以确定。在CS-2OST中也鉴定出了参与与PAPS结合的类似残基。在这两种酶中还鉴定出了参与碳水化合物底物结合的其他残基。这些残基处的突变导致磺基转移酶活性丧失，但保留了结合磷酸腺苷5'-磷酸的能力。HS-2OST的催化功能似乎涉及两个组氨酸残基（His140和His142），而CS 2-OST中可能只有一个组氨酸（His168）至关重要。HS 2-OST催化残基的这一独特特征促使我们对果蝇硫酸乙酰肝素2-O-磺基转移酶进行表征。这项研究的结果为深入了解不同残基在HS-2OST和CS-2OST酶的生物学作用中所起的异同提供了依据。
