Just as humanized antibodies seek to duplicate the amino acid sequence produced in humans, researchers are hot on the heels of “designer” glycoproteins with tailor-made glycans. Since glycosylation cannot be genetically encoded, investigators must resort to artificial means to achieve desirable, glycosylation patterns. “Glycan trimming,” a method employed by Heather Desaire, Ph.D., a chemistry professor at the University of Kansas (Lawrence), may be one step toward glycosylation-by-design.
The subtractive method uses glycosidase enzymes, in Dr. Desaire’s case a-mannosidase, to remove mannose monosaccharides selectively from high-mannose glycans and thereby “expose” desirable sugar residues. Enzymes specific for other sugars may also be used.
“The method is designed so that either an individual monosaccharide type is removed, or, if more than one deglycosylating enzyme is used, multiple monosaccharide types could be trimmed from the glycoprotein,” Dr. Desaire says. After the enzymatic reactions, purification and analysis ensures the desired glycosylation profile was obtained, and that the protein folded properly.
Structure-activity relationships for glycosylation are still in an early stage. Most correlations are empirical. Given that limitation, glycan trimming may be employed to improve circulating half-life, in the case of mannose-trimming, by reducing the opportunity for mannose binding lectin (MBL) to clear glycoproteins from circulation.
“We have not shown experimentally that trimmed proteins have longer circulation lifetimes, but the binding of MBL to high mannose glycoproteins is well known,” comments Dr. Desaire. Other potential benefits are reducing immunogenicity by removing sialic acid and other immunogenic residues, and improving stability or solubility.
For years protein glycosylation was believed to be an exclusive trait of eukaryotes. The discovery, in 1999, that the bacterium Campylobacter jejuni added glycans to proteins, was therefore a startling revelation. Research by Christine Syzmanski (National Research Council of Canada), and Brendan Wren and Rebecca Langdon, Ph.D., a scientist at the London School of Hygiene and Tropical Medicine, among others, has firmly established that bacteria conduct widespread N-glycosylation of proteins.
Dr. Langdon’s group has identified genes implicated in protein glycosylation in several different Campylobacter and Helicobacter species. “While we now have a good understanding of how N-glycosylation functions in C. jejuni, we are still unclear of its role in the biology of the bacterium and also how the system works in the other bacteria where the genes have been identified,” Dr. Langdon says.
Using a technique known as protein-glycan-coupling technology, she has expressed protein-glycosylation genes in E. coli, an organism more familiar to bioprocessors than Campylobacter or Helicobacter. Dr. Langdon is currently developing this system to produce protein-glycoconjugate vaccines.
Other groups, particularly Professor Mario Feldman’s at the University of Alberta are working on O-glycosylation as well. However, with O-glycosylation there is less control over the protein acceptor site. “Using N-glycosylation, we can engineer proteins to include the acceptor site for attachment of the sugar substrate. This creates the opportunity to theoretically link any polysaccharide to any protein.”
The implications for (future) glycoprotein manufacture in simple bacterial systems are tremendous, but for now the near-term potential remains in glycoconjugate vaccines. Dr. Langdon has been collaborating with GlycoVaxyn, to commercialize such vaccines.