Site-specific immobilization of the endosialidase for studying polysialic acid reveals QSOX2 is a novel polysialylated protein

ABSTRACT: 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, in part due to the lack of tools with which to study the glycan. 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 and EndoNDM on streptavidin-coated magnetic or agarose beads. We showed that immobilizing EndoN improves enzyme usefulness by allowing for effective removal of the enzyme from samples, while retaining hydrolase activity. Additionally, immobilization of EndoNDM allowed for the enrichment of polysialylated proteins from complex mixtures for their identification via mass spectrometry. We identified QSOX2 as a novel polysialylated protein secreted from MCF-7 cells. This method of site-specific immobilization can be utilized for other enzymes and lectins to yield insight into glycobiology.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Serum

SUBMITTER: Nichollas Scott  

LAB HEAD: Lisa M. Willis

PROVIDER: PXD046194 | Pride | 2024-04-05

REPOSITORIES: Pride