Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Glycobiology. 2017 Jul 1;27(7):646-656. doi: 10.1093/glycob/cwx021.
Glycosaminoglycans (GAGs) have therapeutic potential in areas ranging from angiogenesis, inflammation, hemostasis and cancer. GAG bioactivity is conferred by intrinsic structural features, such as disaccharide composition, glycosidic linkages and sulfation pattern. Unfortunately, the in vitro enzymatic synthesis of defined GAGs is quite restricted by a limited understanding of current GAG synthases and modifying enzymes. Our work provides insights into GAG-active enzymes through the creation of sulfated oligosaccharides, a new polysaccharide and chimeric polymers. We show that a C6-sulfonated uridine diphospho (UDP)-glucose (Glc) derivative, sulfoquinovose, can be used as an uronic acid donor, but not as a hexosamine donor, to cap hyaluronan (HA) chains by the HA synthase from the microbe Pasteurella multocida. However, the two heparosan (HEP) synthases from the same species, PmHS1 and PmHS2, could not employ the UDP-sulfoquinovose under similar conditions. Serendipitously, we found that PmHS2 co-polymerized Glc with glucuronic acid (GlcA), creating a novel HEP-like polymer we named hepbiuronic acid [-4-GlcAβ1-4-Glcα1-]n. In addition, we created chimeric block polymers composed of both HA and HEP segments; in these reactions GlcA-, but not N-acetylglucosamine-(GlcNAc), terminated GAG acceptors were recognized by their noncognate synthase for further extension, likely due to the common β-linkage connecting GlcA to GlcNAc in both of these GAGs. Overall, these GAG constructs provide new tools for studying biology and offer potential for future sugar-based therapeutics.
糖胺聚糖（GAGs）在血管生成、炎症、止血和癌症等领域具有治疗潜力。GAG 的生物活性取决于内在的结构特征，如二糖组成、糖苷键和硫酸化模式。不幸的是，由于对当前 GAG 合成酶和修饰酶的了解有限，体外酶促合成特定 GAG 的方法受到很大限制。我们的工作通过合成硫酸化寡糖、一种新的多糖和嵌合聚合物，为 GAG 活性酶提供了新的认识。我们表明，C6-硫酸化尿苷二磷酸（UDP）-葡萄糖（Glc）衍生物硫酸奎诺糖可以作为一种戊糖酸供体，但不能作为己糖胺供体，通过来自微生物多杀巴斯德菌的透明质酸（HA）合酶来封闭 HA 链。然而，来自同一物种的两种肝素聚糖（HEP）合成酶，PmHS1 和 PmHS2，在类似条件下不能使用 UDP-硫酸奎诺糖。偶然的是，我们发现 PmHS2 与葡萄糖醛酸（GlcA）共聚 Glc，形成一种我们称为 hepbiuronic 酸 [-4-GlcAβ1-4-Glcα1-]n 的新型 HEP 样聚合物。此外，我们还合成了由 HA 和 HEP 片段组成的嵌段共聚物；在这些反应中，GlcA-，而不是 N-乙酰葡萄糖胺（GlcNAc）-，终止的 GAG 受体被它们非同源的合成酶识别，以进一步延伸，可能是由于这两种 GAG 中 GlcA 与 GlcNAc 之间的共同β 键连接。总的来说，这些 GAG 构建体为研究生物学提供了新的工具，并为未来基于糖的治疗提供了潜力。
