A Novel Technique for Characterizing Polysialylated Proteins from Complex Mixtures

• Author / Creator

  Derksen, Tahlia R L

• Polysialic acid (polySia) is a long homopolymer of sialic acid residues that has profound consequences for the proteins it is attached to. Its expression is restricted in healthy human adults to the nervous, reproductive, and immune systems. It contributes to neuronal development and plasticity along with cell migration and immune suppression. PolySia is dysregulated in a variety of neurological diseases, mental health disorders, autoimmune diseases, and cancers. Abnormally high expression is associated with advanced disease and poor prognosis. However, many of the mechanisms of polySia in both health and disease remain poorly understood. This is partially due to not knowing which proteins are polysialylated and how they contribute to disease progression. As our analyses indicate that there are more polysialylated proteins than the handful documented in known and unknown sources, there is a need for further investigation.
  We have developed a novel technique to identify polysialylated proteins from complex mixtures such as serum or cell lysate. In this method we have biotinylated a polySia lectin to immobilize on streptavidin agarose. The agarose beads can be added to complex mixtures to isolate polysialylated proteins. These isolated proteins undergo protein identification using mass spectrometry. This method improves upon immunoprecipitation techniques by allowing for vigorous washes with detergent.
  The use of this method was validated by correctly identifying neural cell adhesion molecule (NCAM/CD56) as a polysialylated protein in the non-Hodgkin’s lymphoma cell line NK-92. The utility has been further demonstrated by identifying novel polysialylated proteins from primary human T cells and invasive breast ductal carcinoma cell line MCF-7.
  Along with the development of this technique, I have characterized polySia in the rare but fatal rheumatic disease scleroderma. This is the first documentation of polySia dysregulation in scleroderma. We have identified polySia both in scleroderma fibroblasts and serum and my novel technique will lead to the identification of the polysialylated proteins in scleroderma.
  In addition to improving our understanding of the role polySia plays in health and disease, these identified proteins have the potential to be used for diagnostic and prognostic testing. This proteomics method is versatile and will be useful for identifying polysialylated proteins from various sources such as immune cells, cancers, and other diseases.

• Subjects / Keywords

  • Polysialic acid
  • Proteomics
  • Scleroderma

• Graduation date

  Fall 2023

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-jkv3-2p93

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.