Department of Biological Sciences, University of Alberta, 116 St & 85 Ave, Edmonton, AB, T6G 2R3, Canada.
Department of Microbiology and Immunology, University of Melbourne, Melbourne, VIC 3000, Australia.
Glycobiology. 2024 Apr 19;34(5). doi: 10.1093/glycob/cwae026.
Polysialic acid (polySia) is a linear polymer of α2,8-linked sialic acid residues that is of fundamental biological interest due to its pivotal roles in the regulation of the nervous, immune, and reproductive systems in healthy human adults. PolySia is also dysregulated in several chronic diseases, including cancers and mental health disorders. However, the mechanisms underpinning polySia biology in health and disease remain largely unknown. The polySia-specific hydrolase, endoneuraminidase NF (EndoN), and the catalytically inactive polySia lectin EndoNDM, have been extensively used for studying polySia. However, EndoN is heat stable and remains associated with cells after washing. When studying polySia in systems with multiple polysialylated species, the residual EndoN that cannot be removed confounds data interpretation. We developed a strategy for site-specific immobilization of EndoN on streptavidin-coated magnetic beads. We showed that immobilizing EndoN allows for effective removal of the enzyme from samples, while retaining hydrolase activity. We used the same strategy to immobilize the polySia lectin EndoNDM, which enabled the enrichment of polysialylated proteins from complex mixtures such as serum for their identification via mass spectrometry. We used this methodology to identify a novel polysialylated protein, QSOX2, which is secreted from the breast cancer cell line MCF-7. This method of site-specific immobilization can be utilized for other enzymes and lectins to yield insight into glycobiology.
聚唾液酸（polySia）是一种由α2,8-连接的唾液酸残基组成的线性聚合物，由于其在调节健康成年人的神经系统、免疫系统和生殖系统方面的关键作用，具有重要的生物学意义。polySia 在几种慢性疾病中也失调，包括癌症和精神健康障碍。然而，polySia 在健康和疾病中的生物学机制在很大程度上仍然未知。聚唾液酸特异性水解酶神经氨酸酶 NF（EndoN）和无催化活性的聚唾液酸凝集素 EndoNDM 已被广泛用于研究聚唾液酸。然而，EndoN 具有热稳定性，并且在洗涤后仍然与细胞结合。当在具有多种多唾液酸化物种的系统中研究 polySia 时，无法去除的残留 EndoN 会混淆数据解释。我们开发了一种将 EndoN 特异性固定在链霉亲和素包被的磁性珠上的策略。我们表明，固定化 EndoN 允许有效地从样品中去除酶，同时保持水解酶活性。我们使用相同的策略固定聚唾液酸凝集素 EndoNDM，这使得能够从血清等复杂混合物中富集多唾液酸化蛋白，然后通过质谱法对其进行鉴定。我们使用这种方法鉴定了一种新的多唾液酸化蛋白 QSOX2，它从乳腺癌细胞系 MCF-7 中分泌出来。这种特异性固定化方法可用于其他酶和凝集素，以深入了解糖生物学。
