GlycoNet research team uses bacterial enzymes to boost the expression of human polysialyltransferase

GlycoNet research team led by Dr. Warren Wakarchuk (left) and Dr. Stephen Withers (right) uses direct evolution to increase activity of stability of enzymes for clinical applications.

by Ryerson University News Staff

If you take a holiday road trip to Arizona and your car breaks down, the mechanic is going to need to know your location. When asked where to find you, it wouldn’t help much to reply “here at the Grand Canyon.” Yes, a truck could be dispatched, but where exactly would it end up? With almost 4,000 square kilometres to cover, you might get lucky and get your fix, but chances are greater that you won’t.

The same basic idea holds true if you have damaged cells in your body – from a trauma such as a spinal cord injury or a condition like Parkinson’s or Alzheimer’s – and the possibility exists to send stem cells into a localized area to regenerate lost or degraded tissue. The more precise and targeted the migratory cells, the better the transplant therapy.

Such was the challenge taken on by co-authors Bettina Janesch, Department of Chemistry and Biology, Ryerson University, and Lars Baumann, Departments of Chemistry and Biochemistry and Michael Smith Laboratory, University of British Columbia, in their published research in Glycobiology. Under the guidance of project supervisor Warren Wakarchuk of Ryerson University (now at University of Alberta) and Associate Scientific Director of GlycoNet, the team investigated a biochemical way to increase polysialic acid addition onto proteins on the surface of cells to make those cells repel other cells and allow for migration.

“Polysialic acid helps to direct cells to specific sites for more efficient and targeted treatment,” says Ryerson PhD candidate and GlycoNet trainee Sadia Rahmani, one of the contributors to the study. “This has been shown in previous studies. But there is a real challenge in making the polysialyltransferase or PST more effective and available at a faster rate. Basically, we needed a strategy that would allow for rapid polysialic acid production in a controlled manner. That’s where this work is unique.”

Introducing DNA into cells to produce PST is one way of increasing polysialic acid production, but this approach comes with many disadvantages. Instead, the research team used bacterial enzymes, because they are more robust than their human counterparts in modifying different human cell lines. So the team artificially modified the glycosylation of whole cell surfaces by introducing a bacterial enzyme, polysialyltransferase from Neisseria meningitidis (PSTNm), along with the activated sugar donor CMP-sialic acid in their extracellular environment.

The current versions of bacterial enzyme are not optimized for use in clinical applications, being somewhat unstable. The team’s research shows that directed evolution of these enzymes could enhance their activity and stability.

Mapping of the mutations in PST-109 based on a Phyre2 generated model of PSTNm.
Mapping of the mutations in PST-109 based on a Phyre2 generated model of PSTNm.

“Our study revealed that this direct biochemical approach provides a smoother road toward clinical application than the use of gene therapy to boost the expression of human polysialyltransferase,” explains Sadia. “For example, polysialic acid induction on neuronal Schwann cells can significantly enhance their migration, axon growth support, and ability to improve functional recovery after spinal cord injury transplantation.”

Given her background in molecular science and cell biology, Sadia’s contribution to the project was to work with a completely new cell line to demonstrate the utility of polysialyltransferase in modifying cell surfaces by flow cytometry.

“I was surprised that with very few modifications, the assay worked,” she says. “I was pleased to have mastered a new method and demonstrated a new result for the study.”

However, there is still some mystery about what exactly is happening at the cellular level. The team’s next step is to understand and characterize how the polysialylation is changing the organization and activity of surface receptors. This research is being developed as a collaboration between the Wakarchuk Lab for GlycoScience at the University of Alberta and the Antonescu Lab for fundamental and cancer cell biology at Ryerson University.

“We know that cell migration is increased when we use bacterial enzymes for rapid and controlled polysialic acid production, but we don’t know exactly how this happens – just that it does. Next, we’ll work with in vitro cell culture, at the single cell level, to answer this question.”

This article is republished from: Paper of the Month, Faculty of Science, Ryerson University.

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