The influence of glycans on the human gut microbiota is a central topic in contemporary glycoscience. These type of carbohydrates in this environment are not only essential for microbial colonization but also serve important regulatory functions that contribute to immune protection. In this article, we will explore the fundamental roles of glycans, present intriguing case studies, and highlight two practical applications.

Our discussion is informed by the outstanding work of the research team behind a comprehensive study published in May of this year. This study involves scientists from the University of Birmingham and the University of Porto and offers a broad perspective on the current state of the field, addressing key aspects of glycobiology and referencing a wide array of relevant research that has shaped our understanding of the microbiota-glycan connection.

The Glycan Landscape within the Human Microbiota

The collection of lipids and proteins decorated with diverse glycans on the surface of the gut mucosa is called glycocalyx and serves as a major interface between the intestinal lining and microorganisms, as well as with the host’s immune system.  Below, we highlight the key structures within the glycocalyx to illustrate how these carbphydrates shape the gut environment, drive microbial interactions, and influence immune system regulation.

Illustration of a healthy colonic human glycocalyx, from The role of glycans in health and disease: Regulators of the interaction between gut microbiota and host immune system. In Seminars in Immunology (Vol. 73, p. 101891). Academic Press.

• Glycoproteins and Glycolipids: The epithelial cell membrane expresses highly diverse glycoproteins and glycolipids, which are decorated with N-linked and O-linked glycans. These carbohydrates, when recognized by immune cells, can dictate the nature of the immune response—either pro-inflammatory or anti-inflammatory.
• Glycan-Binding Proteins (GBPs): Immune cells produce GBPs, such as galectins, siglecs, and C-type lectins, which «read» the glycan code expressed by both host cells and microbes.
• Glycosaminoglycans (GAGs): Chondroitin sulfate, heparan sulfate, and hyaluronan are also important glycans of the glycocalyx. They serve as nutrient sources for gut microbes, such as Bacteroides thetaiotaomicron, highlighting their role in microbial metabolism and colonic health.
• Mucins (MUC1–22): Mucins form the protective mucus layer overlaying the glycocalyx. As an example, it has been shown that MUC1 functions as a decoy to distract and confuse Helicobacter pylori during infection.
• Carbohydrate-Active Enzymes (CAZymes): Gut microbes produce CAZymes like glycoside hydrolases (enzymes depicted as «scissors») to degrade complex glycans into short-chain fatty acids (SCFAs). SCFAs act as essential nutrients for colonocytes and help maintain gut homeostasis.

The Influence of Glycans: Intriguing Case Studies

Several studies have identified the significant role of certain glycans within our microbiota in regulating immune system responses and determining which microbes colonize our gut, ultimately influencing our health and susceptibility to various diseases.

A noteworthy example of this interaction can be seen in research on infants who are either breastfed or formula-fed. Studies reveal that human milk oligosaccharides (HMOs) play a crucial role in fostering the growth of Bifidobacterium longum subsp. infantis, a beneficial bacterium that thrives predominantly in the gut of breastfed infants.

In a more surprising twist, some pathogens have evolved a strategy known as glycan mimicry, where they display glycans on their surface that immune cells fail to recognize as harmful, allowing them to evade detection. A striking example is Neisseria gonorrhoeae, the bacteria responsible for gonorrhea, which incorporates sialic acid from the host onto its lipooligosaccharides, effectively camouflaging itself from the immune system.

Targeting the Glyco-Microenvironment: Two Practical Applications

Several approaches are being explored to target the intestinal glyco-microenvironment, including the enhancement of the mucosal glycome through glycan supplementation or by modifying the activity of CAZymes. Two recent studies have made significant strides in translating these concepts into practical applications.

One study, published in Nature, involves researchers from Washington University School of Medicine and the International Centre for Diarrhoeal Disease Research in Dhaka, Bangladesh. The team identified key bioactive glycan structures, contributing to the development of their MDCF-2 project. This therapeutic food is designed to help restore the gut microbiota of malnourished children, offering hope for improving their gastrointestinal health.

Another study, published in the Proceedings of the National Academy of Sciences, focuses on how gut microbes process plant-derived N-glycans. By identifying specific enzymes that can modify and analyze these N-glycan structures, the research aims to apply these findings to medical and industrial fields. One of the possible applications is to reduce the risk of allergic reactions to foods and medications by leveraging this knowledge.

Conclusions

The intricate interactions between glycans and gut microbes are crucial for understanding and maintaining gut health, as well as for treating immune diseases and gastrointestinal disorders. At Asparia Glycomics, we specialize in supporting cutting-edge research in this field. Whether you’re developing HMOs for synthetic milk or unraveling the composition and structure of bioactive oligosaccharides and polysaccharides, our expertise in glycan synthesis and analysis is here to guide you. We have a proven track record of assisting projects in gut microbiota research and development.

As advances in glycomics continue to unfold, they are unlocking transformative medical applications, from restoring microbiomes in malnourished children to mitigating allergic reactions through glycan engineering. These special carbohydrates undoubtedly hold the key to groundbreaking solutions in human health, and we at Asparia Glycomics are ready to partner with you on this exciting journey. Reach out to explore how we can support your innovation.

References

• Crouch, L. I., Rodrigues, C. S., Bakshani, C. R., Tavares-Gomes, L., Gaifem, J., & Pinho, S. S. (2024, May). The role of glycans in health and disease: Regulators of the interaction between gut microbiota and host immune system. In Seminars in immunology(Vol. 73, p. 101891). Academic Press.
• Crouch, L. I., Urbanowicz, P. A., Baslé, A., Cai, Z. P., Liu, L., Voglmeir, J., … & Bolam, D. N. (2022). Plant N-glycan breakdown by human gut Bacteroides. Proceedings of the National Academy of Sciences, 119(39), e2208168119.
• Hibberd, M. C., Webber, D. M., Rodionov, D. A., Henrissat, S., Chen, R. Y., Zhou, C., … & Gordon, J. I. (2024). Bioactive glycans in a microbiome-directed food for children with malnutrition. Nature, 625(7993), 157-165.
• Wang, Y., Ze, X., Rui, B., Li, X., Zeng, N., Yuan, J., … & Li, M. (2021). Studies and application of sialylated milk components on regulating neonatal gut microbiota and health. Frontiers in Nutrition, 8, 766606.

The quest to improve cancer diagnostics and therapeutics remains at the forefront of modern science, and glycoscience may hold the potential for groundbreaking progress. Why focus on glycoscience? Because changes in glycosylation—a fundamental post-translational modification—are closely associated with cancer progression. These glycosylation alterations often lead to unique glycan structures that can serve as reliable biomarkers for various cancers. Indeed, glycan-based biomarkers have been identified in several cancer types, including breast, ovarian, colorectal, prostate, pancreatic cancers, as well as lymphoma and myeloma, promising to enhance early detection and personalized treatment approaches.

But how close are we to fully implementing these findings to transform patient care? What advancements have already been made, and which techniques show the most promise? In this article, we delve into the current landscape of glycan-focused cancer diagnostics and therapeutics, spotlight two recent studies with compelling results, and share insights from Antoine Lesur, analytical scientist and Head of Analytics at Asparia Glycomics.

The Need for Standardization and Multidisciplinary Efforts

The well-established link between aberrant glycosylation and the initiation and progression of numerous cancers has propelled cancer glycomics research towards identifying specific glycan biomarkers. These biomarkers hold the potential to revolutionize cancer diagnosis and personalized treatment. Over the past few decades, efforts have largely focused on advancing technologies that allow the observation of large-scale glycosylation changes. Additionally, considerable progress has been made in connecting glycans to their protein carriers and exploring how these modifications impact intracellular signaling and cellular functions.

Despite these advancements, we are still at the early stages of fully deciphering glycosylation’s complex role in cancer. One of the critical challenges hindering successful biomarker discovery is the lack of standardized and accessible protocols for glycan sample preparation, data acquisition, and analysis. Therefore, collaborative efforts with experts across pertinent fields are essential. As Antoine Lesur emphasizes, «During the steps of biomarker discovery, verification, and validation, the choice of samples must be made in advance with the help of clinicians and statisticians to ensure that the data will be relevant.»

In addition, inter-subject variability studies should be integrated into glycan biomarker research. As outlined in our publication «Mass Spectrometry for Glycan Biomarker Discovery,» to ensure accuracy, any glycan biomarker study must include an analysis on variability between the genetic and demographic differences among individuals and due to specific pathological conditions.

Mass Spectrometry Techniques Driving the Present and Future

Various glycan analysis techniques can be applied to biomarker discovery, with mass spectrometry (MS) being an indispensable tool due to its precision in mass measurement for compositional analysis and its ability for structural identification.

For N- glycan analysis, a commonly employed MS technique is MALDI (matrix-assisted laser desorption/ionization), where a sample on a solid support can be combined with a matrix and undergo ionization. This technique is applied to the analysis of tissues of clinical samples in cancer.

For classic glycomics, it is also common to integrate complementary techniques. Mass spectrometry methods allow the possibility to couple with separation techniques like liquid chromatography, capillary electrophoresis and ion mobility. These combined techniques offer significant potential for separating isomers, which could be crucial for discovering new biomarkers. They are also regularly employed for quality control in biotherapeutics used in the treatment of cancer.

Breakthrough Studies of 2024 in Cancer Glycomics Research

In May of this year, researchers from Ivanov Lab at Northeastern University published a study in Nature Communications reporting unique results. They successfully applied capillary electrophoresis followed by mass spectrometry to analyze a single still-living human cell. Before this, nobody had examined, measured, and determined the structure of whole proteins and native glycans from an individual cell. This marked a relevant advanced towards diagnostics tests for a variety of diseases, including cancer.

Just a month later, a team from the Australian Institute for Bioengineering and Nanotechnology introduced another innovative technology in Advanced Science, that lead to significant results. They developed a nanodevice capable of analyzing glycopatterns of small extracellular vesicles (sEVs) released by lung cancer cells. In a clinical study of 40 patients, this nanotechnology distinguished early-stage malignant lung nodules from benign ones, demonstrating the potential of profiling small EV glycans for noninvasive lung cancer diagnostics and prognostics.

Tumor-Associated Carbohydrate Antigens: Potential Cancer Biomarkers

Among the diverse range of cancer biomarkers, Tumor-Associated Carbohydrate Antigens (TACAs) have drawn attention from researchers: «TACAs are glycan structures that are abnormally expressed in cancer cells. They play significant roles in tumor growth, metastasis, and immune evasion, which makes them potential biomarkers for cancer diagnosis and potential therapeutic targets», explains Antoine.

Some common TACAs are Thomsen-nouvelle antigen (Tn), sialyl Thomsen-nouvelle antigen (STn) and sialyl Lewis antigens. Tn and STn antigens have been identified in nearly all types of human carcinomas and are highly overexpressed in breast, colon, lung, pancreatic, and prostate cancers.

TACAs are commonly identified using chromatographic techniques combined with mass spectrometry. This process starts with extracting glycoproteins from serum or plasma, followed by purification involving antibodies or lectins, and then the glycans are detached from the peptide backbone through chemical or enzymatic treatments. Afterwards, the glycans are separated based on their structure polarity and size, allowing direct analysis via native MS.

Conclusions

Significant progress has been made with analytical techniques like mass spectrometry and the study of tumor-associated carbohydrate antigens (TACAs). However, challenges remain—particularly around standardization and the need for a stronger multidisciplinary approach.

To truly position glycoscience as a cornerstone of personalized medicine, greater investment, specialized expertise, and precision are essential. Asparia’s Glycan Analysis Service, led by Antoine Lesur, supports this vision by offering precise mapping of glycosylation patterns, enabling the accurate identification and synthesis of TACAs to propel your research and development forward.

References

• Aizpurua-Olaizola, O., Toraño, J. S., Falcon-Perez, J. M., Williams, C., Reichardt, N., & Boons, G. J. (2018). Mass spectrometry for glycan biomarker discovery. TrAC Trends in Analytical Chemistry, 100, 7-14.
• Doud, E. H., & Yeh, E. S. (2023). Mass spectrometry-based glycoproteomic workflows for cancer biomarker discovery. Technology in Cancer Research & Treatment, 22, 15330338221148811.
• Marie, A. L., Gao, Y., & Ivanov, A. R. (2024). Native N-glycome profiling of single cells and ng-level blood isolates using label-free capillary electrophoresis-mass spectrometry. Nature Communications, 15(1), 3847.
• Puiu, M., Nativi, C., & Bala, C. (2023). Early detection of tumour-associated antigens: Assessment of point-of-care electrochemical immunoassays. TrAC Trends in Analytical Chemistry, 160, 116981.
• Zhou, Q., Niu, X., Zhang, Z., O’Byrne, K., Kulasinghe, A., Fielding, D., … & Trau, M. (2024). Glycan Profiling in Small Extracellular Vesicles with a SERS Microfluidic Biosensor Identifies Early Malignant Development in Lung Cancer. Advanced Science, 2401818.