Site-specific immobilization of the endosialidase reveals QSOX2 is a novel polysialylated protein

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,...

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Bibliographic Details
Published in:Glycobiology (Oxford) 2024-04, Vol.34 (5)
Main Authors: Hunter, Carmanah, Derksen, Tahlia, Makhsous, Sogand, Doll, Matt, Perez, Samantha Rodriguez, Scott, Nichollas E, Willis, Lisa M
Format: Article
Language:English
Subjects:
Adult
Humans
Lectins
Neuraminidase - metabolism
Oxidoreductases Acting on Sulfur Group Donors
Sialic Acids - chemistry
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Summary: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.
ISSN:1460-2423
1460-2423
DOI:10.1093/glycob/cwae026