Why Glycans?
Background
In addition to DNA, proteins and lipids, glycans are one of the four major components of a cell. Glycans are involved in almost all basic functions of multicellular organisms and are found on the dense outer layer of membrane proteins and lipids attached to the glycocalyx. This glycocalyx is comprised of multiple types of complex carbohydrates (glycoproteins, proteoglycans, hyaluronic acid, glycolipids) on the outer layer of the cell surface that are used by the cell to indicate “self” vs. “non-self” to the immune system and activate the innate immune system response mechanisms when necessary.
Modifications in the glycocalyx must occur in order to allow for cell mobility and migration. One of the major changes that occurs is the post-translational modification of glycosylation of proteins. Glycosylation is the enzymatic process that involves the sequential addition of individual carbohydrates to proteins and lipids.
Functions
The biological functions of glycans can be broken into 4 main categories:
structural/modulatory functions
intrinsic (intraspecies) recognition
extrinsic (interspecies) recognition
molecular mimicry/appropriation of host glycans
The glycome is unique because its formation combines both genetic and environmental influences. The glycome is influenced by genetics because of the proteins involved in glycan synthesis, and it is also influenced by environmental factors that are integrated at the level of glycan biosynthesis.
N-Glycans
N-linked glycans are oligosaccharides that are covalently bonded to proteins at asparagine residues through an N-glycosidic bond. Most cell surface receptors are N-glycosylated, including integrins, transforming growth factor β receptor (TGFβ), and EGFR. Modified N-glycans have the ability to affect protein folding, protein stability, carbohydrate-carbohydrate interactions, carbohydrate-protein interactions, and glycoprotein-glycoprotein interactions.
Modified oligosaccharides are able to regulate a variety of physiological and pathological processes. Changes in N-linked glycosylation are of particular interest because they have been linked to cell mobility, cell growth, intracellular signaling, metastatic capacity, and cellular immune properties. Further understanding and study of the structure and functions of N-glycans and their associated glycoproteins will reveal new mechanistic insights to disease states, potential therapeutic targets and prognostic tools.
Representative examples of N-glycan localization to specific cortex and medulla regions in a normal kidney tissue.
Figure from Drake, R. et. al. J. Mass Spectrom. 2020.
Glycans, Proteoglycans, Glycoproteins and Protein-Binding Glycans in the Hallmarks of Cancer
Figure based on Peixoto, A. et.al. Front. Oncol. 2019.
Glycans, proteoglycans, glycoproteins and protein-binding glycans located on the exterior of the plasma membrane drive the interaction between cancer cells and the tumor microenvironment (a complex scaffold of extracellular matrix and other cell types). This has been proven through decades of research identifying specific alternations in glycosylation that directly impact all stages of disease and contribute to more aggressive cell phenotypes. The biosynthesis of cancer-associated glycans and their influence in the glycoproteome is driven by microenvironmental cues that act synergistically towards disease evolution.
These intricate interactions lay the molecular foundation for the activation of relevant oncogenic pathways and result in functional alterations that stimulate invasion and drive disease. The glycoproteome and the tumor microenvironment contribute to the progression of cancer (the hallmarks of cancer) by influencing cell adhesion, cell-cell recognition, intracellular signaling and extracellular matrix interactions.
n-glycan imaging applications
Serum/Plasma Profiling
A novel slide-based N-glycan profiling method was evaluated for sensitivity and reproducibility. Using this method, over 75 N-glycan species can be detected from one microliter of serum in less than 6.5 hours, demonstrating the potential applications of this method in clinical diagnostics.
Profiling of Cultured Cells
Image from Angel, P. et. al. J. Proteome Res. 2019
Typically, N-glycosylation studies done on cultured cells can require up to millions of cells followed by lengthy preparation to release, isolate, and profile N-glycans. To overcome these limitations, a rapid array-based workflow for profiling N-glycan signatures from cells was developed. This method was adapted from imaging mass spectrometry (IMS) used for on-tissue N-glycan profiling. Using this approach, N-glycan profiles from a low-density array of 8 cell chambers could be reported within four hours of completing cell culture.
Antibody Captured Glycoprotein Profiling
Antibody panel based N-glycan imaging is a novel platform for N-glycan analysis of immunocaptured proteins. This platform consists of antibodies spotted in an array panel to a microscope slide, specific capture of glycoproteins from a biological sample, and then enzymatic release of N-glycans for analysis by MALDI MSI. N-glycans are detected at each individual spot, allowing N-glycan information to easily be linked back to its protein carrier.
Glycosylation Analysis from FFPE Tissues
Mapping the two-dimensional distribution of N-glycans in tissues is possible through the use of MALDI MSI. The method is capable of spatially profiling the location and distribution of multiple N-linked glycan species released by PNGase F in frozen or formalin-fixed tissues. This method can be applied to tissue or tumor microarrays to analyze changes in glycosylation in countless disease states.