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Glycosylation in cellular mechanisms of health and disease

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Cells and cell populations function in a concerted manner through the communication between various macromolecules and subcellular compartments. Glycans are one of the main elements of cooperation in cellular mechanisms. Their vastly diverse roles include protein folding and stability, signal transduction, intracellular protein trafficking, cell differentiation, and immune response.

In the same breath, aberrant glycosylation can result in abnormal glycan structures with devastating effects. Disruptions in mechanisms of cell development, differentiation, signaling, and division are all recipes for some of the most hard-to-master diseases. In particular, rare congenital disorders and cancers possess disease phenotypes that are hard to fathom by simply looking at genetic mutations. To understand how cellular mechanisms are tweaked in the first place, we need to take a closer look at the glycan attached to the essential proteins that orchestrate homeostasis. 

Here we provide an overview of the role of glycans in healthy cell machinery as well as examples of altered glycans in congenital diseases and cancer. 

Glycans in Healthy Cellular Mechanisms 

The main cellular functions of glycans can be divided into three classes (you can look at our previous blog post for an introduction to the universe of glycans): structural/modulatory roles, intrinsic recognition, and extrinsic recognition. The structural/modulatory roles of glycans encompass a series of functions that help maintain cell integrity, membrane organization, and diffusion while shielding the cell through protective barriers.

Intrinsic recognition is central to the transport of nutrients as well as intracellular protein trafficking for signaling and degradation. Finally, extrinsic recognition involves robust pathogen identification and immune response by recognizing pathogen-specific glycans. Examples of these functions can be seen more evidently in specific cell types. 

One of the most noticeable cases is the role of glycans in neural function. In particular, N-glycans were found to be significant for the transmission of electric impulses and chemical neuromodulators.

Research shows that sialylated N-glycans are primary components of voltage-gated ion channels and are necessary for axon firing (1). Additionally, they are of central importance in the proper function of glial cells responsible for supporting neurons and establishing homeostasis in the central nervous system. More specifically, proteins that are responsible for glutamate transport (2) and blood-brain barrier formation (3) are N-glycosylated. 

Another case in point involves platelets responsible for blood clotting. Recent studies unveiled the role of glycosylation in platelet production and count. Animal studies show that the absence of glycosyltransferases, such as ST3Gal4 (4) and β4GalT1 (5), could lead to low platelet counts, indicating the importance of sialylation for platelet production. Even after platelet production, the importance of glycosylation remains, as the lack of sialylation was shown to cause premature platelet clearance (6).  

The role of glycans in hormonal balance is yet another area of exploitation and can unravel their influence on many physiological processes, from appetite to anxiety and alertness. A close look at biologically active peptide hormones reveals that one-third of peptide hormones identified in the human body are O-glycosylated. More importantly, these glycopeptides are necessary for facilitating receptor interactions and extending peptide half-lives (7). 

The Disease of Glycosylation: Altered Cellular Function 

Through examples from various specialized cell types, the role of glycosylation in a functioning cell became evident. When we turn the table, we also find that aberrant glycosylation is a driving factor in the onset of rare diseases and cancers. 

Congenital Disorders of Glycosylation 

Congenital Disorders of Glycosylation are a specific set of hereditary disorders that modify glycosylation networks. They are often caused by the absence of one or more enzymes. The Genetic and Rare Diseases Information Center (GARD) has identified 19 such diseases while indicating that approximately 50,000 people in the U.S. suffer from them. 

GALNT2 is a gene encoding for the member of glycosyltransferases that initiate O-glycosylation in mucin-type peptides that are of central importance to tissue development and metabolism. Among the many functions of this gene, the regulation of high-density lipoprotein cholesterol (HDL-C) stands out.

Often called “good” cholesterol, HDL is responsible for the transfer of low-density lipoprotein—a.k.a. bad—cholesterol (LDL-C) to the liver, thus mediating its clearance. Patients with low HDL-C levels have an increased propensity for heart disease and stroke due to LDL-C accumulation.

Gene knockout studies revealed a striking relationship between GALNT2 and HDL-C levels. In mice harboring GALNT2 loss-of-function, the phospholipid transfer protein responsible for LDL-C uptake from tissues was less active (8).

Another possible consequence of GALNT2 deficiency was recently revealed. Loss-of-function studies in mice helped identify a novel congenital disorder characterized not only by low HDL-C but also by intellectual disability, impaired cognitive function, epilepsy, and developmental defects, among many other debilitating symptoms (9). The effect of GALNT2 mutations on neurodevelopment continues to be investigated.  

GALNT3 is a similar protein-encoding gene that gives rise to UDP-GalNAc transferase 3, necessary for O-glycosylation initiation. A sequence analysis study from 2004 showed that the deletion of this gene was highly associated with a rare metabolic disorder called familial tumoral calcinosis (FTC) (10).

Individuals suffering from FTC often possess abnormal phosphate and calcium deposits around joints, severely hindering joint movement. Studies also showed that GALNT3 deficiency resulted in the inhibition of the glycosylation of fibroblast growth factor 23, which is responsible for calcium and phosphate transfer between tissues and the kidney.  

GALNT2 and GALNT3 are two examples of severe cases where the absence of a particular glycosyltransferase can manifest in devastating ways in the body.  

Cancer and Glycosylation 

The role of glycosylation in cancer cannot be overstated. The variety of genetic mutations aside, the cancerous phenotype is also driven by the mechanistic changes inside the cell and in the extracellular matrix (ECM). Research shows that changes in the surface glycan content are associated with increased stiffness of the cell and the extracellular matrix that biases the cells’ mechanotransduction pathways into cancerous phenotype (11). This improves the ability of cancer cells to proliferate, differentiate, adhere to ECM, and migrate.   

Proteoglycans seem to possess a prominent role in mediating the cancer response to mechanical cues from the ECM. For example, an increased level of heparan sulfate proteoglycans (HSPGs), such as agrin, promotes the assembly of substrate adhesion molecules that attach cancer cells to the ECM (12). Another transmembrane HSPG called syndecan-4 was shown to change conformation in response to mechanical stiffening, promoting the binding of scaffold proteins and activating the kindlin-integrin-RhoA pathway involved in adhesion to surrounding tissues (13). 

Transmembrane proteins, such as mucin, are heavily glycosylated. Mucin is a critical component of cellular integrity, as it often serves as a protective barrier. Research suggests a staggering increase of truncated O-glycans in cell surface MUC1 and MUC16. It is revealed that altered glycosylation changes the surface distribution of mucins. In healthy cells, mucins are sequestered from receptor tyrosine kinases, and their interactions are limited.

Through aberrant glycosylation, mucins get uniformly distributed throughout the entire cell surface and come into contact with kinases more frequently, which results in the overactivation of kinases and their downstream binding partners. Furthermore, this redistribution makes cancer cells resistant to degradation (14).  

You can look at the SpeakEasy blog to learn more about the specific downstream effects of glycan alterations in the tumor microenvironment and in cancer progression.  

Conclusion 

Protein glycosylation patterns can greatly determine the mechanistic properties and dynamic behavior of cells. Abnormalities can drive malignant behavior, such as uncontrolled signal transduction, immune checkpoint evasion, and ECM-mediated metastasis. That’s why profound strategies are required to study glycosylation at the single-cell level. An emerging technique is the application of transcriptomics to investigate cellular glycosylation.

One such platform is Glycopacity, which can run single-cell RNA-seq to identify 214 glycosylation-related enzymes and their RNA-level regulations (15). In addition, Surface-protein Glycan and RNA sequencing (SUGAR-seq) integrates glycosylation profiles and single-cell RNA-seq data, empowering discovery of novel biology. This method was employed to identify unique glycan epitopes in tumor-infiltrating T cells (16). These breakthroughs can pave the way for understanding the role of glycosylation in health and disease more precisely. 

With the help of research initiatives, our understanding of glycosylation will continue to expand. To that end, the National Institutes of Health (NIH) initiated a Glycoscience program to develop reliable glycobiology resources, assay kits, and high-throughput screening tools to assist researchers. The program also aims to develop integrative data analysis tools to make glycan analysis more streamlined.  

Last but not least, our SpeakEasy Science Blog and Glycobiology eBook serve as scientific hubs where you can find practical information about glycan detection and quantification as well as reviews of peer-reviewed articles exploring the roles of glycans in health and disease.  

References

  1. Kruger LC, et al. 2016. Voltage-Gated Na+ Channels: Not Just for Conduction. Cold Spring Harbor Perspectives in Biology. 
  2. Bauer D, et al. 2010. Abnormal Glycosylation of EAAT1 and EAAT2 in Prefrontal Cortex of Elderly Patients With Schizophrenia. Schizophrenia Research
  3. Jing B, et al. 2018. Glycosylation of Dentin Matrix Protein 1 is a Novel Key Element for Astrocyte Maturation and BBB Integrity. Protein & Cell
  4. Grozovsky R, et al. 2014. The Ashwell-Morell Receptor Regulates Hepatic Thrombopoietin Production via JAK2-STAT3 Signaling. Nature Medicine. 
  5. Giannini S, et al. 2020. β4GALT1 Controls β1 Integrin Function to Govern Thrombopoiesis and Hematopoietic Stem Cell Homeostasis. Nature Communications. 
  6. Greenberg J, et al. 1975. Effects on Platelet Function of Removal of Platelet Sialic Acid by Neuraminidase. Laboratory Investigation; A Journal of Technical Methods and Pathology. 
  7. Madsen TD, et al. 2020. An Atlas of O-linked Glycosylation on Peptide Hormones Reveals Diverse Biological Roles. Nature Communications
  8. Khetarpal SA, et al. 2016. Loss of Function of GALNT2 Lowers High-Density Lipoproteins in Humans, Nonhuman Primates, and Rodents. Cell Metabolism. 
  9. Zilmer M, et al. 2020. Novel Congenital Disorder of O-linked Glycosylation Caused by GALNT2 Loss of Function. Brain. 
  10. Topaz O, et al. 2004. Mutations in GALNT3, Encoding a Protein Involved in O-Linked Glycosylation, Cause Familial Tumoral Calcinosis. Nature Genetics. 
  11. Purushothaman A, et al. 2023. The Role of Glycans in the Mechanobiology of Cancer. Journal of Biological Chemistry
  12. Chakraborty S, et al. 2015. An Oncogenic Role of Agrin in Regulating Focal Adhesion Integrity in Hepatocellular Carcinoma. Nature Communications. 
  13. Chronopoulos A, et al. 2020. Syndecan-4 Tunes Cell Mechanics by Activating the Kindlin-Integrin-RhoA Pathway. Nature Materials. 
  14. Kufe DW. 2012. MUC1-C Oncoprotein as a Target in Breast Cancer: Activation of Signaling Pathways and Therapeutic Approaches. Oncogene. 
  15. Dworkin LA, et al. 2022. Applying Transcriptomics to Study Glycosylation at the Cell Type Level. iScience. 
  16. Kearney CJ, et al. 2021. SUGAR-seq Enables Simultaneous Detection of Glycans, Epitopes, and the Transcriptome in Single Cells. Science Advances
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Shuhui Chen, PhD