Immunoglobulin G (IgG) antibody molecule with glycan attached. Inset shows glycan structure. Credit: RCSB Protein Data Bank
Realizing the dream of truly personalized medicine is a huge goal in the biomedical research landscape. Biomarker discovery is a fundamental arm of progress in personalized medicine that has been revolutionized by advances in exploring genomic, proteomic and metabolomic markers of health and disease. But one promising sector remains largely unexplored: glycomic biomarkers, which encompass the glycans and patterns of glycosylation present in the body.
Glycans are complex, branched chains of polysaccharides and oligosaccharides that join to proteins and lipids in a process called glycosylation. Their presence throughout cells and tissues and their role in maintaining normal biological functions—from facilitating vital cell-to-cell interactions to guiding protein folding—make glycans an ideal potential class of biomarkers for numerous diseases. Research in detecting and characterizing dysregulated glycosylation and its impact on downstream processes will be a key contribution to the development of new diagnostics and therapeutics.
Pamela James, Vice President of Vector Laboratories, recently spoke to Labcompare about the importance of glycans, technologies to advance glycan research and how glycobiology is posed to impact future health and disease research.
Q: The glycosylation process has been notoriously difficult to study. What new technologies are helping to make screening more scalable and consistent to achieve reliable results?
A: Mass spectrometry (MS) has long been employed to analyze glycan content but the limited sensitivity to low-abundance glycan populations was a challenge. In recent decades, a variety of methods have been developed for to enhance glycan sample preparation, enrichment, and separation prior to the use of MS. Many of these methods also incorporate fluorescent detection, allowing greater separation and identification of glycan populations with better reproducibility. These approaches are generally not as high throughput but eliminate the complication of pre-ionization competition. The use of MALDI-TOF-MS and enhanced derivatization of glycans, including specific glycan linkages, have further advanced our ability to detect differences in glycosylation between healthy and diseased tissues. This approach allows more high-throughput analysis. A limitation of these studies is that they are typically done with serum or extracted tissue samples that then lose spatial context. Mass spectrometry imaging with MALDI has also made recent advances in mapping N-glycan differences. Additional techniques such as MALDI-2 are making headway to becoming quantitative analytical glycomics techniques but are still plagued by variabilities that require diligent experimental control and sample preparation. While many of these MS-related techniques have been used for years, they are not commonplace laboratory techniques and require highly trained personnel to run experiments and perform data analysis. This often requires a laboratory studying glycobiology to send samples to specialized core lab. The paper “High-Throughput Glycomic Methods” recently published in Chemical Reviews by Trbojević-Akmačić, et al has a great overview. There is a plethora of similar papers published in the last 4 years that describe more recent advances in high-throughput glycan analysis.
Q: What other instruments and equipment are vital to glycosylation research?
A: Another approach being explored is to apply proteomics methodologies to glycan analysis using lectins. Lectins can be described as proteins having affinity for specific glycan structures, much like antibodies have affinity for protein epitopes. While researchers have used lectins for over 40 years, their utilization in proteomics platforms is becoming more common, perhaps driven by increasing interest in glycosylation. Lectins have been employed in methods like CyTOF and proximity ligation assays. Adaptation of using lectins in these platforms can be relatively straightforward as long as a researcher is aware the incorporation of lectins requires a thoughtful approach. Many of the primary antibodies and blocking reagents used in these platforms are also glycosylated and can lead to off-target binding. All of the typical protein analytical approaches can be applied to lectins, and many labs already have the necessary laboratory instruments and equipment. Western blotting, ELISA, flow cytometry, etc. can be easily adopted for glycan analysis.
Because of the complexity of the data that comes from some glycan analytics like glycan arrays, advances in software and databases have furthered the utility of these types of studies. There are many papers published detailing these advances. The NIH Common Fund has also been supporting a Glycoscience program for years that is dedicated to advancing the tools, technologies, and resources to make the study of glycans accessible to all researchers.
Q: What has existing research on glycosylation in health and disease told us?
A: It’s becoming increasingly clear that glycosylation has a role in almost all biological functions. We haven’t even scratched the surface of understanding those interactions or how they can be utilized to detect disease and influence disease outcomes.
Q: What should near-future research address in glycan discovery? Are there any other diseases/conditions that you think the study of glycosylation can help?
A: Because glycosylation is so ubiquitous across biological systems, it’s relevant to the study of virtually every disease or phenomenon. Right now, I’d say the most prominent areas for studying glycosylation are cancer, infectious disease, and autoimmunity. Glycans are a very promising class of oncology biomarkers, as they regulate interactions between tumor and immune cells and are implicated in tumor cell signaling, migration, and proliferation. Because glycans play such a vital role in the immune system’s ability to distinguish “self” vs. “other,” they’re also very important in immune-pathogen interaction as well as chronic inflammatory and autoimmune disorders.
Q: Glycans are complex. In glycobiology research, why is it so important to understand the factors behind glycan structural diversity?
A: Without fully understanding the diversity of glycans, we’re missing another layer of information on how biological systems function and what drives dysfunction. It would be incredibly powerful to be able to tie in this layer of knowledge with what we’ve learned from genomics, transcriptomics, and proteomics. Glycosylation is among the most common post-translational modifications—imagine how limited we would be right now if scientists hadn’t recognized the power of protein phosphorylation or if the world of genomics didn’t grasp the impact of epigenetic modification.
Q: How can scientists and research advocates help make glycobiology more mainstream?
A: First, by recognizing that glycosylation almost assuredly plays a role in the system or disease one is studying. Second, by teaming up with those who are studying glycobiology or glycomics and combining that expertise with that of immunologists, cancer biologists, neuroscientists, or virologists to gain more insight into the dynamics of that biological system. Collaboration and sharing of knowledge can further understanding in any field of study. Funding glycobiology sessions in the context of other conferences like AACR and AAI is also important to ensuring that more researchers are exposed to the impacts of glycobiology. At the Society for Glycobiology conference, many of the attendees and presenters did not start their research careers in glycobiology but in other fields. Creating the opportunity for those researchers to interface and share their work with others in their same field can accelerate the adoption of glycan analysis.
Pamela James, Ph.D., serves as Vice President, Product, for Vector Laboratories where she oversees research and development and leads quality assurance, product, and program management. James has spent over a decade and a half with Vector Laboratories in multiple scientific and director roles. James led the introduction of several impactful products from Vector Laboratories and is focused on Vector’s mission to bring glycobiology tools to the broader scientific community. She earned a Ph.D. in Immunology from UMass Chan Medical School and a B.S. in Biochemistry from California Polytechnic State University-San Luis Obispo.