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A Guide to Glycan-Binding Proteins

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The human body is home to a multitude of glycans attached to different macromolecules in various locations in the cell and within the cellular environment. This mind-blowing diversity makes the complete analysis of glycans challenging and calls for robust glycan-recognizing probes.

Fortunately, the last 2 decades witnessed the discovery of macromolecules with useful glycan-binding features, such as plant lectins, antibodies, and proteins with carbohydrate-binding modules (CBMs). These tools can be isolated and utilized with many characterization techniques. Furthermore, advancements in synthetic biology are paving the way for more diverse and sensitive probes.

Classification of Glycan-Binding Proteins

Lectins

Lectins are a broad class of proteins known for their ability to bind carbohydrates. While the endogenous lectins interact with glycans to mediate several essential biological processes, lectins from other sources have an equally important quest: detecting and quantifying glycans.

Plant Lectins

Plant lectins are currently the gold standard for lectin-based analyses. Many plant lectins are abundant in nature and easy to extract. Furthermore, they display highly specific and reversible carbohydrate-binding activity, which makes them versatile glycan recognition tools. They have been employed in glycan analysis of various cancers, viral diseases, and autoimmune disorders (1).

Recent research went a step further and used plant lectins as therapeutics and for enhanced targeting of cancer and infectious diseases (2). You can gain invaluable insight into the various applications of plant lectins in detection and treatment by taking a look at the SpeakEasy science blog.

Recombinant Lectins

Despite their wide adoption in glycan analysis, isolating plant lectins in pure forms and high biomasses remains a challenge. Synthetic biology, particularly recombinant DNA technology, has been proposed for tackling these challenges. Researchers use a range of host cells, including bacteria, yeast, and mammalian cells, to express tailor-made plant lectins using lectin cDNA libraries. The resulting recombinant lectins are reported to have less contamination and less batch-to-batch variation than their natural counterparts, driving better accuracy and reproducibility.

Oliveira et al. provided a comprehensive review of strategies and applications involving recombinant lectins, highlighting the advantages and disadvantages of every host (3).

Improving Natural Lectin Specificity

​​In the meantime, several other methods have been established for enhancing the natural lectin specificity towards glycans. Site-directed mutagenesis involves inducing a mutation in 1 or more amino acid residues to augment specificity.

Another option is to apply randomized mutations to create a large lectin library. One study employed mutagenesis to Peanut agglutinin by mutating its Asn41 residue, which enhanced its specificity towards the tumor-associated TF antigen (Galβ1−3GalNAc) (4). Another study endowed galactose binding to a naturally mannose-binding lectin by mutagenesis at multiple residues (5). ​

Besides mutating the existing binding site, adding new binding sites to a lectin has been explored. These lectins became multivalent, significantly improving their binding affinities to the respective glycans (6).

Antibodies

Another class of glycan-recognizing probes stems from our immune system, produced by B cells through repeated exposure to foreign substances, especially microbes. These endogenous glycan-binding antibodies serve critical protective functions by recognizing surface glycans of pathogenic bacteria, viruses, and fungi. Other endogenous antibodies are responsible for recognizing tumor-associated carbohydrate antigens and can trigger an immune response.

Recombinant Antibody

A newly emerging field in anti-glycan antibodies is the use of recombinant DNA technology. More specifically, one can generate a large library of antibody-encoding genes, which are then introduced into host cells to yield the set of antibodies. The resulting antibody library can subsequently be used to select and amplify the antibodies with the highest affinity towards the antigen of interest.

The recombinant antibody technology can be particularly beneficial for maximizing the efficacy of antibodies. For example, Amon et al. used a yeast surface display to design an antibody library against sialyl Lewis a (SLea) for the treatment of colon and pancreatic cancers (7). The library of mutant antibodies displayed higher cytotoxicity towards SLea-positive cancer cells than the native SLea antibody.

Researchers from Harvard Medical School followed a similar approach to generate an antibody-encoding gene library from sea lamprey, which yielded much higher glycan specificity than conventional mouse monoclonal antibodies.

Carbohydrate-Binding Modules (CBMs)

Carbohydrate-active enzymes are responsible for building or degrading complex carbohydrates. Research revealed that these enzymes carry 1 or more domains called carbohydrate-binding modules (CBMs that can target the enzyme to the polysaccharide by recognizing specific carbohydrates.

Today, protein engineering enables heterologous expression of CBMs in large quantities. Thus, an intriguing alternative to antibodies and lectins is the use of synthetic biology to engineer CBMs for glycan recognition purposes.

Characterization of Glycan-Binding Proteins (GBP)

The proper characterization of glycan-binding proteins is necessary for the applications mentioned above to succeed. Researchers should evaluate and confirm the binding characteristics of proteins, especially in the case of engineered proteins.

Surface Plasmon Resonance (SPR) and titration calorimetry are the most widely used methods of GBP characterization without disrupting molecular structures (8).

Surface Plasmon Resonance (SPR)

SPR involves immobilizing the glycan of interest on a metal surface and measuring glycan binding through mass changes deduced from the electromagnetic oscillation frequency of the surface electrons. It allows a real-time estimate of the binding interaction, which provides quantitative insight, such as association and dissociation constants. However, SPR requires sufficient glycan attachment to the surface for precise measurements.

Isothermal Titration Calorimetry (ITC)

Isothermal titration calorimetry (ITC estimates glycan binding from changes in heat induced by binding, which helps deduce enthalpy, entropy, and binding constants. It has particularly been the preferred method for measuring binding affinities in mutant/altered GBPs and comparing their binding properties to the wild types. While it eliminates the need for glycan immobilization, it is not suitable for high-throughput characterization since an ITC chamber can take 1 GBP-glycan interaction at a time.

Glycan Microarray

One can generate a better high-throughput readout using glycan microarrays comprising various simple and complex carbohydrates immobilized on a plate. This allows the simultaneous characterization of multiple lectins or antibodies for binding specificity.

What makes glycan microarrays stand out amongst other methods is the availability of large glycan microarray libraries and toolkits curated from experimental data, enabling visualization and analysis of glycan microarray datasets. Among the most widely-used analysis platforms of glycan microarray datasets are CarboGrove and GlycoToolKit – Glycan Array Dasboard (GLAD). Also, Haab et al. elaborates on many other resources and step-by-step implementation of glycan microarrays (9).

Applications of Glycan-Binding Proteins

Each of the glycan-recognizing probes described above has distinct advantages that help boost glycan analysis applications, including but not limited to antibody- or lectin-based microarrays, immunoassays (e.g., ELISA), cell sorting, purification of glycoconjugates or glycans for further analysis, glycosyltransferase kinetic assays, immunofluorescence (IF), and immunohistochemistry (IHC).

Each of these applications uncovers different insights about glycobiology. Glycan and glycoprotein purification through affinity chromatography help identify post-translational protein modifications and prevalent glycan epitopes on these proteins. This can be beneficial when determining the mutant glycans on altered and diseased samples. On the other hand, IHC/IF approaches help characterize surface glycoconjugates, revealing their cellular locations, spatial orientations, and relative abundance in cells.

More recent approaches involve cloning glycosyltransferase genes. For example, researchers can identify novel genes encoding specific glycosyltransferases and transfect cell lines with those genes to characterize their functions. Enzyme function, quantity, and activity can also be deduced using lectins and antibodies.

Lectin Applications

Lectins are widely used as tumor biomarker detectors, where they detect the unique glycan epitopes present in malignant cancer cells. This helps distinguish cancer subtypes more accurately, determining the stage, metastatic potential, and aggressiveness. Lectin microarray consists of a panel of lectins and it is the preferred strategy for accelerated high-throughput biomarker discovery.

For example, lectin microarrays were used to characterize triple-negative breast cancer (TNBC), which constitutes 15% of breast cancer cases and is characterized by its metastatic and treatment-resistant nature. Using lectin microarrays on 6 TNBC cell lines, researchers identified Ricinus communis agglutinin I as the lectin that could detect the aberrant surface glycans on TNBC (10).

Lectins can be incorporated into IHC/IF applications to understand the glycosylation state to visualize and analyze tumor microenvironment as well as disease characteristics such as cancer progression and metastatic behavior. Shuhui Chen, PhD and Erika Leonard, PhD from Vector Laboratories applied a set of plant lectins to different cancerous tissues to characterize differential glycosylation states across cancer types. Their work demonstrated the utility of plant lectins for robust cancer biomarker discovery.

Besides diagnostic potential, many plant lectins exhibit anti-cancer activity. One of them is a mannose-binding lectin from Pinellia pedatisecta that triggers cancer cell death in lung cancer and hepatocellular carcinoma (11). Another mannose- and glucose-specific lectin isolated from the seeds of Dioclea sclerocarpa intervened in cancer cell cycles at immune checkpoints in ovarian and prostate cancer (12).

Glycan-Binding Antibody Applications

Besides their intrinsic functions, endogenous anti-glycan antibodies contribute to diagnostics. By quantifying antibodies in patient serum, researchers develop tools to diagnose bacterial infections (13), autoimmune diseases (14), and cancer (15).

The anti-glycan antibody production machinery has also been exploited for vaccine production. Carbohydrate-based vaccines can activate immune response by introducing immunogenic glycans for the body to generate antibodies. Many glycan-based vaccines have been FDA-approved and widely used since the 90s for the prevention of influenza (16), meningitis (17), and pneumonia (17).

Cancer vaccine research is another exciting area that studies glycans to activate the immune response to cancer cells. These vaccines mainly comprise tumor-associated carbohydrate antigens (TACAs), including but not limited to the Tn, TF, and sialyl-Tn antigens (18).

Although these glycans induce weak immunogenicity on their own, this can be overcome by coupling the antigens to other immunogenic molecules, such as carrier proteins. Thus, the vaccine can direct T cell-dependent immune responses to the TACAs (19). There are currently 4 cancer vaccines in phase 3 clinical trials for breast cancer (20–21), non-small cell lung cancer (22), and melanoma (23).

Besides prevention, glycan-binding antibodies have also gained recognition for treatment. Researchers can now synthesize monoclonal antibodies in large quantities for treating infectious diseases and cancer (24). These antibodies act either by binding to cancer cell surface glycans for easier recognition by the immune system or by blocking the glycoproteins responsible for dampening the checkpoint-based immune response.

Carbohydrate-binding Modules (CBMs) Applications

CBMs have been used in glycan identification. Sialidases, responsible for cleaving terminal sialic acid residues, are among the enzymes under investigation. Their sialic acid recognition mechanism, combined with recombinant technology, can help design robust sialic acid-recognizing tools.

One great example is the work of Ribiero et al., who engineered a sialic acid CBM from Clostridium perfringens, which showed higher affinity to α(2,3)-sialyl-lactose than any other natural lectins in the same class. The research team also engineered a dimeric version of the CBM with an added affinity towards 6’-sialyl-lactose (24).

Conclusion

The discovery and development of novel glycan-binding probes is on the rise. Consequently, their applications can diversify and evolve our understanding of glycobiology and its role in health and disease.

Understanding the basics of glycobiology and glycan recognition is fundamental for state of the art glycan analysis. Our SpeakEasy blog is a great place to start, as it contains several informative articles about the basics of glycobiology and applications of glycan-recognizing probes such as lectins. You can also find publication reviews demonstrating the applications of lectins in glycobiology.

In addition, Essentials of Glycobiology is a practical resource for learning the basics of carbohydrates and enhancing your knowledge of advanced glycobiology research.

Lastly, the Alliance of Glycobiologists for Cancer Research, established by the National Cancer Institute, provides a list of additional resources, including glycan databases, projects by the Consortium for Functional Glycomics, and symposiums.

References

  1. Bah CSF, et al. 2013. Medicinal Applications of Plant Lectins. Antitumor Potential and other Emerging Medicinal Properties of Natural Compounds.
  2. Katoch R, et al. 2021. Research Advances and Prospects of Legume Lectins. Journal of Biosciences.
  3. Carla Oliveira JA, et al. 2012. Recombinant Lectins: An Array of Tailor-Made Glycan-Interaction Biosynthetic Tools. Critical Reviews in Biotechnology.
  4. Adhikari P, et al. 2001. Mutational Analysis at Asn-41 in Peanut Agglutinin: A Residue Critical for the Binding of the Tumor-Associated Thomsen-Friedenreich Antigen. Journal of Biological Chemistry.
  5. Drickamer K. 1992. Engineering Galactose-Binding Activity Into a C-Type Mannose-Binding Protein. Nature.
  6. Yabe R, et al. 2009. Engineering a Versatile Tandem Repeat-Type α2-6sialic Acid-Binding Lectin. Biochemical and Biophysical Research Communications.
  7. Amon R, et al. 2020. Directed Evolution of Therapeutic Antibodies Targeting Glycosylation in Cancer. Cancers.
  8. Warkentin R, et al. 2021. Resources and Methods for Engineering “Designer” Glycan-Binding Proteins. Molecules.
  9. Haab BB, et al. 2020. Advances in Tools to Determine the Glycan-Binding Specificities of Lectins and Antibodies. Molecular & Cellular Proteomics.
  10. Zhou SM, et al. 2015. Lectin RCA-I Specifically Binds to Metastasis-Associated Cell Surface Glycans in Triple-Negative Breast Cancer. Breast Cancer Research.
  11. Lu Q, et al. 2012. Pinellia Pedatisecta Agglutinin Interacts With the Methylosome and Induces Cancer Cell Death. Oncogenesis.
  12. Li Ping, et al. 2017. Caspase-9: Structure, Mechanisms and Clinical Application. Oncotarget.
  13. Seow CH, et al. 2009. Novel Anti-Glycan Antibodies Related to Inflammatory Bowel Disease Diagnosis and Phenotype. American Journal of Gastroenterology.
  14. Brettschneider J, et al. 2009. Serum Anti-GAGA4 IgM Antibodies Differentiate Relapsing Remitting and Secondary Progressive Multiple Sclerosis From Primary Progressive Multiple Sclerosis and Other Neurological Diseases. Journal of Neuroimmunology.
  15. Tikhonov A, et al. 2020. Glycan-Specific Antibodies as Potential Cancer Biomarkers: A Focus on Microarray Applications. Clinical Chemistry and Laboratory Medicine.
  16. Ahonkhai VI, et al. 1990. Haemophilus Influenzae Type B Conjugate Vaccine (Meningococcal Protein Conjugate) (PedvaxHIB): Clinical Evaluation. Pediatrics.
  17. Goldschneider I, et al. 1969. Human Immunity to the Meningococcus: I. The Role of Humoral Antibodies. Journal of Experimental Medicine.
  18. Temme JS, et al. 2021. Anti-Glycan Antibodies: Roles in Human Disease. Biochemical Journal.
  19. Jin KT, et al. 2019. Recent Advances in Carbohydrate-Based Cancer Vaccines. Biotechnology Letters.
  20. Miles D, et al. 2011. Phase III Multicenter Clinical Trial of the Sialyl‐TN (STn)‐Keyhole Limpet Hemocyanin (KLH) Vaccine for Metastatic Breast Cancer. The Oncologist.
  21. Huang CS, et al. 2016. Randomized Phase II/III Trial of Active Immunotherapy with OPT-822/OPT-821 in Patients With Metastatic Breast Cancer. Journal of Clinical Oncology.
  22. Segatori VI, et al. 2018. Antibody-Dependent Cell-Mediated Cytotoxicity Induced by Active Immunotherapy Based on Racotumomab in Non-Small Cell Lung Cancer Patients. Cancer Immunology, Immunotherapy.
  23. Kirkwood JM, et al. 2016. High-Dose Interferon Alfa-2b Significantly Prolongs Relapse-Free and Overall Survival Compared With the GM2-KLH/QS-21 Vaccine in Patients With Resected Stage IIB-III Melanoma: Results of Intergroup Trial E1694/S9512/C509801. Journal of Clinical Oncology.
  24. Ribeiro JP, et al. 2016. Characterization of a High-Affinity Sialic Acid-Specific CBM40 From Clostridium Perfringens and Engineering of a Divalent Form. Biochemical Journal.
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Shuhui Chen, PhD