Bidondo Lucía, Landeira Mercedes, Festari Florencia, Freire Teresa, Giacomini Cecilia
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.
Biochem Biophys Rep. 2021 Mar 2;26:100940. doi: 10.1016/j.bbrep.2021.100940. eCollection 2021 Jul.
Sialic acids are widely distributed in nature and have biological relevance owing to their varied structural and functional roles. Immobilized neuraminidase can selectively remove terminal N-acetyl neuraminic acid from glycoproteins without altering the protein backbone while it can be easily removed from the reaction mixture avoiding sample contamination. This enables the evaluation of changes in glycoprotein performance upon desialylation.
Neuraminidase was immobilized onto agarose activated with cyanate ester groups and further used for desialylation of model glycoproteins, a lysate from tumour cells and tumour cells. Desialylation process was analysed by lectin binding assay, determination of sialyl-Tn or flow cytometry.
neuraminidase was immobilized with 91 % yield and expressed activity yield was of 41%. It was effective in the desialylation of bovine fetal serum fetuin, bovine lactoferrin and ovine submaxilar mucin. A decrease in sialic-specific SNA lectin recognition of 83% and 53 % was observed for fetuin and lactoferrin with a concomitant increase in galactose specific ECA and PNA lectin recognition. Likewise, a decrease in the recognition of a specific antibody (82%) upon mucin desialylation was observed. Moreover, desialylation of a protein lysate from the sialic acid-rich cell line TA3/Ha was also possible leading to a decrease in 47 % in SNA recognition. Immobilized neuraminidase kept 100% of its initial activity upon five desialylation cycles.
Immobilized neuraminidase is an interesting as well as a robust biotechnological tool for enzymatic desialylation purposes.
Immobilized neuraminidase would contribute to understand the role of sialic acid in biological processes.
唾液酸在自然界广泛分布，因其多样的结构和功能作用而具有生物学相关性。固定化神经氨酸酶可选择性地从糖蛋白中去除末端N - 乙酰神经氨酸，而不改变蛋白质主链，同时可轻松从反应混合物中去除，避免样品污染。这使得能够评估去唾液酸化后糖蛋白性能的变化。
将神经氨酸酶固定在经氰酸酯基团活化的琼脂糖上，进而用于模型糖蛋白、肿瘤细胞裂解物和肿瘤细胞的去唾液酸化。通过凝集素结合试验、唾液酸-Tn测定或流式细胞术分析去唾液酸化过程。
神经氨酸酶的固定化产率为91%，表达活性产率为41%。它对牛胎血清胎球蛋白、牛乳铁蛋白和羊下颌粘蛋白的去唾液酸化有效。观察到胎球蛋白和乳铁蛋白对唾液酸特异性SNA凝集素的识别分别降低了83%和53%，同时半乳糖特异性ECA和PNA凝集素的识别增加。同样，观察到粘蛋白去唾液酸化后特异性抗体的识别降低了82%。此外，对富含唾液酸的细胞系TA3/Ha的蛋白质裂解物进行去唾液酸化也是可行的，导致SNA识别降低了47%。固定化神经氨酸酶在五个去唾液酸化循环后保持其初始活性的100%。
固定化神经氨酸酶是一种用于酶促去唾液酸化目的的有趣且强大的生物技术工具。
固定化神经氨酸酶将有助于理解唾液酸在生物过程中的作用。
