含 6-磷酸甘露糖 (M6P) 的糖蛋白配体的化学酶合成和受体结合揭示了 M6P 受体识别的异常结构要求,Journal of the American Chemical Society

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含 6-磷酸甘露糖 (M6P) 的糖蛋白配体的化学酶合成和受体结合揭示了 M6P 受体识别的异常结构要求
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2016-08-16 , DOI: 10.1021/jacs.6b05762
Takahiro Yamaguchi ₁ , Mohammed N. Amin _{1,

2} , Christian Toonstra ₂ , Lai-Xi Wang _{1,

2}

Affiliation

6-磷酸甘露糖 (M6P) 末端寡糖是 M6P 受体介导的将新合成的水解酶从高尔基体靶向溶酶体的重要信号，但由于 M6P 配体受体识别的精确结构要求尚未完全了解难以获得均质的含 M6P 的糖蛋白。我们在这里描述了携带天然 M6P 的 N-聚糖的均质磷酸糖蛋白的化学酶促合成。该方法包括化学合成具有不同数量和位置的 M6P 部分的聚糖恶唑啉，并通过内切糖合酶将它们转移到 GlcNAc 蛋白，以提供均质的含 M6P 的糖蛋白。还完成了两个 M6P-寡糖与环状多肽的同时连接以产生二价 M6P-糖肽。表面等离子体共振结合研究表明，位于低聚甘露糖环境的低 α-1,3-分支的单个 M6P 部分足以与受体 CI-MPR 高亲和力结合，而在 α 处存在 M6P 部分-1,6-分支是可有可无的。此外，对二价环状和线性多肽的结合研究表明，两个 M6P-寡糖配体的紧密接近对于实现对 CI-MPR 受体的高亲和力至关重要。总之，本研究表明 M6P 部分的位置和价态以及正确的寡糖环境对于与主要 M6P 受体的高亲和力结合都是至关重要的。

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Chemoenzymatic Synthesis and Receptor Binding of Mannose-6-Phosphate (M6P)-Containing Glycoprotein Ligands Reveal Unusual Structural Requirements for M6P Receptor Recognition

Mannose-6-phosphate (M6P)-terminated oligosaccharides are important signals for M6P-receptor-mediated targeting of newly synthesized hydrolases from Golgi to lysosomes, but the precise structural requirement for the M6P ligand-receptor recognition has not been fully understood due to the difficulties in obtaining homogeneous M6P-containing glycoproteins. We describe here a chemoenzymatic synthesis of homogeneous phosphoglycoproteins carrying natural M6P-containing N-glycans. The method includes the chemical synthesis of glycan oxazolines with varied number and location of the M6P moieties and their transfer to the GlcNAc-protein by an endoglycosynthase to provide homogeneous M6P-containing glycoproteins. Simultaneous attachment of two M6P-oligosaccahrides to a cyclic polypeptide was also accomplished to yield bivalent M6P-glycopeptides. Surface plasmon resonance binding studies reveal that a single M6P moiety located at the low α-1,3-branch of the oligomannose context is sufficient for a high-affinity binding to receptor CI-MPR, while the presence of a M6P moiety at the α-1,6-branch is dispensable. In addition, a binding study with the bivalent cyclic and linear polypeptides reveals that a close proximity of two M6P-oligosaccharide ligands is critical to achieve high affinity for the CI-MPR receptor. Taken together, the present study indicates that the location and valency of the M6P moieties and the right oligosaccharide context are all critical for high-affinity binding with the major M6P receptor. The chemoenzymatic method described here provides a new avenue for glycosylation remodeling of recombinant enzymes to enhance the uptake and delivery of enzymes to lysosomes in enzyme replacement therapy for the treatment of lysosomal storage diseases.

更新日期：2016-08-16

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