Researchers from Arizona State University's Biodesign Institute have assembled an expression vector library of the enzymes necessary to create, modify, and degrade glycans. They say this library will be a transformative resource for recombinant enzyme production, enabling structure–function studies and expanding applications of these enzymes.
Results of the new study, which were published in Nature Chemical Biology today, have potentially broad impact in areas ranging from new diagnostics and therapeutics for disease, to other advances in health, materials science, and energy, according to Joshua LaBaer, director of the Biodesign Institute and Virginia G. Piper Center for Personalized Diagnostics and co-author on the paper.
"Studying glycan structures in animal cell systems has historically been a big challenge, especially in terms of technology," says Kelley Moremen from the University of Georgia, lead author of the new study. "In order to understand how these molecules are made and regulated in terms of function at the cell surface, it's important to understand the enzymatic machinery that makes them, modifies them, and breaks them down."
Glycans typically attach to specific locations on proteins, modulating their biological activity through molecular recognition or affecting their circulation time in the bloodstream. Glycosylation is one of the most important regulatory mechanisms affecting proteins after they have already been translated from RNA.
"With the help of Jason (Steele) and Josh (LaBaer), we created a design strategy to capture these coding regions and put them in a holding vector," Moremen says. "Then, we were able to slot them into insect or mammalian cells and test their ability to be expressed in those systems."
Previous efforts to produce and express glycoenzymes in simpler bacterial cells like E. coli had largely run aground, according to the team. To manage the feat in more complex eukaryotic cells, like those of insects and mammals, considerable modification of the enzyme coding genes was required before their insertion into specialized vectors known as cassettes. These glycoenzyme-coding cassettes were then inserted into insect and mammalian cells, where they were expressed.
The group produced a comprehensive list of 339 glycoenzymes responsible for formation, modification, and degradation of glycans, targeting these for protein expression. While the results showed successful, high-level expression of glycoenzymes in both insect and mammalian cells, the authors note distinct differences in specific expression levels in each of the two model systems.
The resulting comprehensive library of glycoenzymes provides a vital resource for future advances in glycobiology and is available to researchers worldwide through DNASU.
Image: Glycans or polysaccharides adorn cell surfaces and are important for cell communication, immune response and many other vital physiological processes. A new study assembles a library of enzymes responsible for building and modifying glycans. Image courtesy of Jason Drees, Biodesign Institute at ASU.