André Sabine, Kozár Tibor, Schuberth Ralf, Unverzagt Carlo, Kojima Shuji, Gabius Hans-Joachim
Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians-University Munich, Veterinärstrasse 13, D-80539 Munich, Germany.
Biochemistry. 2007 Jun 12;46(23):6984-95. doi: 10.1021/bi7000467. Epub 2007 May 12.
Core fucosylation and the bisecting N-acetylglucosamine residue are prominent natural substitutions of the N-glycan core. To address the issue of whether these two substituents can modulate ligand properties of complex-type biantennary N-glycans, we performed chemoenzymatic synthesis of the respective galactosylated and alpha2,3/6-sialylated N-glycans. Neoglycoproteins were then produced to determine these glycans' reactivities with sugar receptors in solid-phase assays and with tumor cells in vitro as well as their in vivo biodistribution profiles in mice. Slight protein-type-dependent changes were noted in lectin binding, including adhesion/growth-regulatory galectins as study objects, when the data were related to properties of N-glycans without or with only one core substituent. Monitoring binding in vitro revealed cell-type-dependent changes. They delimited the ligand activity of this glycan type from that of chains with un- and monosubstituted cores. A markedly prolonged serum half-life was conferred to the neoglycoprotein by the galactose-terminated N-glycan, which together with increased organ retention of all three neoglycoproteins underscores the conspicuous relevance for glycoengineering of pharmaproteins. The predominant presentation of the two branches in the disubstituted N-glycan as extended (alpha1,3-antenna) and backfolded (alpha1,6-antenna) forms, revealed by molecular dynamics simulations, can underlie the measured characteristics. These results obtained by a combined strategy further support the concept of viewing N-glycan core substitutions as non-random additions which exert a modulatory role on ligand properties. Moreover, our data inspire us to devise new, non-natural modifications to realize the full potential of glycoengineering for diagnostic and therapeutic purposes.
核心岩藻糖基化和平分型 N-乙酰葡糖胺残基是 N-聚糖核心显著的天然取代基。为了解决这两个取代基是否能调节复合型双天线 N-聚糖的配体特性这一问题，我们对各自的半乳糖基化和 α2,3/6-唾液酸化 N-聚糖进行了化学酶促合成。然后制备新糖蛋白，以在固相分析中确定这些聚糖与糖受体的反应性、在体外与肿瘤细胞的反应性以及它们在小鼠体内的生物分布概况。当数据与没有或只有一个核心取代基的 N-聚糖特性相关时，在凝集素结合中发现了轻微的蛋白质类型依赖性变化，包括以粘附/生长调节半乳糖凝集素作为研究对象。体外监测结合情况发现了细胞类型依赖性变化。它们界定了这种聚糖类型与具有未取代和单取代核心的链的配体活性。半乳糖末端的 N-聚糖赋予新糖蛋白显著延长的血清半衰期，所有三种新糖蛋白在器官中的保留增加，这突出了糖蛋白糖工程的显著相关性。分子动力学模拟显示，双取代 N-聚糖中两个分支的主要呈现形式为伸展型（α1,3-天线）和回折型（α1,6-天线），这可能是所测特性的基础。通过联合策略获得的这些结果进一步支持了将 N-聚糖核心取代视为非随机添加物的概念，这些添加物对配体特性发挥调节作用。此外，我们的数据激励我们设计新的非天然修饰，以实现糖工程在诊断和治疗目的方面的全部潜力。
