Laboratorio de Bioquímica, Departamento de Biociencias, Facultad de Química, UdelaR, Gral. Flores 2124, Montevideo, Uruguay.
Laboratorio de Inmunomodulación y desarrollo de Vacunas, Departamento de Inmunobiología, Facultad de Medicina, UdelaR, Gral Flores 2125, Montevideo, Uruguay.
Biotechnol Appl Biochem. 2022 Feb;69(1):209-220. doi: 10.1002/bab.2099. Epub 2021 Jan 22.
The elucidation of glycans biological function is essential to understand their role in biological processes, both normal and pathological. Immobilized glycoenzymes are excellent tools for this purpose as they can selectively release glycans from glycoproteins without altering their backbone. They can be easily removed from the reaction mixture avoiding their interference in subsequent experiments. Here, we describe the immobilization of peptide-N-glycosidase F (PNGase F) onto silica magnetic nanoparticles with immobilization yields of 86% and activity yields of 12%. Immobilized PNGase F showed higher thermal stability than its soluble counterpart, and could be reused for at least seven deglycosylation cycles. It was efficient in the deglycosylation of several glycoproteins (ribonuclease B, bovine fetuin, and ovalbumin) and a protein lysate from the parasite Fasciola hepatica under native conditions, with similar performance to that of the soluble enzyme. Successful deglycosylation was evidenced by a decrease in specific lectin recognition of the glycoproteins (40%-80%). Moreover, deglycosylated F. hepatica lysate allowed us to confirm the role of parasite N-glycans in the inhibition of the lipopolysaccharide-induced maturation of dendritic cells. Immobilized PNGase F probed to be a robust biotechnological tool for deglycosylation of glycoproteins and complex biological samples under native conditions.
糖链生物学功能的阐明对于理解它们在正常和病理生物过程中的作用至关重要。固定化糖酶是实现这一目标的极好工具，因为它们可以选择性地从糖蛋白中释放糖链，而不改变其骨架。它们可以很容易地从反应混合物中去除，避免它们在后续实验中的干扰。在这里，我们描述了将肽-N-糖基化酶 F（PNGase F）固定到硅胶磁性纳米颗粒上，固定化产率为 86%，活性产率为 12%。固定化 PNGase F 表现出比可溶性对应物更高的热稳定性，并且可以至少重复使用七次进行去糖基化。它在几种糖蛋白（核糖核酸酶 B、牛胎球蛋白和卵清蛋白）和寄生虫 Fasciola hepatica 的蛋白裂解物的天然条件下的去糖基化中非常有效，性能与可溶性酶相似。糖蛋白（40%-80%）对特定凝集素识别的降低证明了成功的去糖基化。此外，去糖基化的 F. hepatica 裂解物使我们能够证实寄生虫 N-聚糖在抑制脂多糖诱导的树突状细胞成熟中的作用。固定化 PNGase F 被证明是一种在天然条件下对糖蛋白和复杂生物样品进行去糖基化的强大生物技术工具。
