The Importance of Lectin and Glycan Binding Specificity

Scientific research is constantly evolving. As new applications, tools, and methodologies emerge and expand, the way scientists approach how to tackle questions and create solutions changes. New mindsets and focuses can lead to new insights within fields. One such field is glycobiology.  

Glycobiology is a well-established yet rapidly expanding field that explores the role of glycosylation. It has a wide array of applications, from tumor biology to drug discovery, allowing scientists to learn more about the importance of glycans as they relate to biological processes in our bodies. A key to utilizing these glycans to unlock deeper insights is lectins. These ubiquitous proteins serve a vast range of purposes in biology through their ability to recognize and bind glycans, making them crucial as we learn how we can leverage glycans for various biotechnology applications to design cutting-edge therapeutics. In introducing lectins into new and established fields and technologies, scientists can open the doors to new discoveries.   

In this blog post, we will walk you through what lectins are, how they bind to glycans, why this process is important, how to understand this process. We will also provide a list of the specificity of certain lectins you can use in your research. Keep reading to learn all this and more, and be sure to check out our Glycan Binding Infographic as well so you can learn how to apply lectins to help your research in new and exciting ways. 

Lectins and glycans  

Lectins are proteins that bind carbohydrate structures (glycans) and are found within numerous plant and animal tissues and organisms. The presence of at least one noncatalytic domain allows them to reversibly recognize and bind to specific carbohydrates without altering their molecular properties (1). As a result, lectins are a valuable tool for biological research with various applications such as virology, cancer, neuroscience, and immunology.  

From the simplest single-celled organisms to humans, all cell types are densely covered with layers of glycans (the collective name for oligosaccharides and polysaccharides) attached to surface proteins, lipids, and as recently discovered, even RNA. These structures facilitate cell-cell interactions, molecular transfer across the cell membrane, cell signaling, and determination of cell fate. Post-translational modifications, such as glycosylation, play a critical role in a myriad of cellular functions. As the product of glycosylation, glycoconjugates are responsible for vital cellular functions from proliferation to immune response. Lectins can be used to discover specific glycan expression patterns that have biological implications such as identifying cell types. Other advantages of plant lectins include their wide availability, ease of extraction, stability, and, of course, their glycan-specific nature. 

How do lectins bind to glycans​? 

Lectins bind glycans at their active site, which is called the carbohydrate recognition domain (CRD). Lectin-glycan interactions do not always involve the entirety of glycan structures. Instead, lectins tend to recognize motifs, specific glycan sequences of usually 1–4 sugars. 

Lectin binding is noncovalent, meaning it does not involve irreversible bond formation. The main interactions involved in lectin binding are hydrogen bonds, van der Waals, and hydrophobic forces (2). 

Direct hydrogen bonding involves the hydroxyl group (OH) of the acidic side chain of the sugar as the acceptor and amide groups (NH)n of CRD residues, mainly asparagine and glutamine. A less common hydrogen bonding that occurs between the sugar -OH and the -OH group of tyrosine, serine, and threonine can also be formed. Hydrogen bonding patterns are useful in determining lectin specificity. For example, GNL/GNA has been determined as a Mannose-specific lectin, as it can form specific hydrogen bonds between the 2’-OH of mannose, while other lectins, such as ConA and pea lectin are not able to form such specific interactions to facilitate tight and specific binding (3).  

A weaker type of hydrogen bonding common to lectin-glycan interactions is water-mediated hydrogen bonds. In some instances, lectins will have water molecules bound naturally in the CRD domain. These water molecules can form hydrogen bond bridges between glycans and lectins to strengthen and support direct hydrogen bonds. 

Glycan chains with charged groups, such as sialic acid (NeuNAc), form hydrogen bonds with sialic acid-binding lectins. Here, the negatively charged carboxylate group (COO) of sialic acid can interact with main chain amide groups, polar side chains, or the water molecules at the lectin’s CRD. If they form with a lysine at the active site, an ionic bond is used. 

Another significant discovery from lectin binding studies is that many lectins, particularly legume lectins, require the presence of metal ions, often divalent cations such as Ca2+ and Mn2+, for binding. In various studies, the removal of cations from the lectin-glycan binding medium abolished their binding properties (4). It is thought that although metal ions do not directly interact with the glycans, they alter the folding of lectins and stabilize them to bring their CRD closer to the glycan epitope, which facilitates binding (5). 

Despite being highly polar molecules, carbohydrates can have nonpolar interactions with lectins and are the drivers of glycan binding as hydrogen bonding cannot drive binding, just shape specificity. Interactions can take place between aromatic side chains of lectins, such as phenylalanine, tryptophan, and tyrosine, and the epimeric center of a carbohydrate. More specifically, epimers are glycan isomers, whose configurations differ at only one carbon. The most well-known example of epimers is D-glucose and D-galactose. Aromatic side chains of lectins can also exhibit nonpolar interactions with the methyl group of the acetamido moieties on GlcNAcGalNAc, and NeuNAc. 

Lectins do not undergo significant conformational changes upon sugar binding, except for the amino acids in the CRD. Subtle differences across glycan motifs can lead to significant changes in the positioning of amino acids in the CRD, impacting the types of interactions that can occur. This aspect of binding is the core mechanism that leads to the differential recognition of sugars. 

Why is binding specificity important? 

Specific lectin binding patterns have been leveraged to purify and isolate glycans and glycan conjugates. Lectins have also been used to identify and quantify glycans in biological samples to reveal their abundance or changes in diseased tissues. Furthermore, through immunohistochemistry and immunofluorescence, these lectins can be monitored during the progression of cellular activities. The applications of lectins have promising implications for early diagnosis and targeted therapies. 

As effective as plant lectins are, they have not reached their full potential. This is in part due to the fact that we have not been able to fully dissect and comprehend the machinery responsible for achieving such high binding specificities. Although lectin inhibition assays and crystallization methods can help recognize oligosaccharides and disaccharides, they only characterize a limited number of glycan structures. With a better understanding of how lectins bind to complex and biologically relevant glycan epitopes, we can extend the reach of lectins into biomedical applications. 

According to Lara Mahal, PhD, Canada Excellence Research Chair in Glycomics at the University of Alberta, understanding the glycan-binding specificity of lectins and how it is achieved is important for a multitude of reasons. “Using lectins to understand biology is predicated on understanding what is bound. For example, knowing that SNA binds specifically a-2,6 sialylation allows us to identify this change in the development of pancreatic cancer, which we can then tie back to the enzymes that create this epitope (ST6GAL1). Without that intimate knowledge of specificity, the binding information loses its value.” She goes on to explain that “If we understand how binding specificity works, we may be able to engineer even more specific lectins in the future, tuning them to bind new epitopes not covered by our current binders.” 

As far as the value this all brings to scientific research, Dr. Mahal believes that As our understanding of binding specificity for the lectins deepens, it will allow us to go beyond superficial annotations of glycan structure. For example, if a group of glycans with different terminal binding (i.e. sialylation, fucosylation, etc.) share a common underlying motif (e.g. type II LacNAc), loss of this underlying structure could cause changes in all binding.” Mahal says, “Knowing this would allow us to focus on the appropriate structures and related enzymes when examining the biology.”    

How to understand glycan-binding specificity​ 

Glycan array technology is one of the pioneering ways to study lectin specificity. In this method, glycans are purified and immobilized on a glass slide. Then, dye-labeled lectins are incubated on these surfaces, where they localize to the sites of the glycans they recognize. After incubation, the amount of lectin attached to each glycan can be quantified by measuring fluorescent signal intensity to determine which glycans can be bound by a specific lectin.  

Glycan arrays help researchers study the preferential binding of lectins to different glycan motifs. With advanced glycan data management software, such as GlycoSearch, it is possible to illustrate cases where lectins recognize multiple motifs (6). Such software tools can compare the binding affinity of a lectin to multiple glycans and rank motifs according to lectin preference. 

Machine learning tools can be combined with glycan array analysis to enhance our scope of knowledge. This is especially necessary to cover as many biologically relevant glycan motifs as possible. Machine learning models can be trained with glycan sequences as inputs and lectin binding motifs as outputs. Such algorithms provide an opportunity to predict lectin binding specificity as well as the conditions for tolerance and inhibition. 

Specificity of Lectins 

Mahal et al. provides a comprehensive appendix on binding patterns for 57 unique plant lectins (7). From this study, we have learned not only the glycan sequences recognized by these lectins but also how binding specificity is impacted by chemical changes in the glycan structure. This can help explain why the same lectin exhibits differential binding affinity to different forms of the same glycan chain. 

Mannose Binding Lectins 

High mannose epitopes are important target epitopes in neutralizing antibody response to human immunodeficiency virus (HIV) (8). The Glc3Man9GlcNAc2 structure gets trimmed by oligosaccharyltransferases to form the epitope Man7-Man9. Although we can encounter modified mannose structures on noncanonical O-glycans, they are predominantly found on N-glycans. Lectins with specificity towards high mannose epitopes are as follows: 

Galanthus Nivalis Lectin (GNA, GNL) 

  • Preferential binding towards α1,3 mannose epitopes 
  • Unlike other mannose-binding lectins, it does not bind α-linked glucose 
  • Binds α2-macroglobulin in human serum 
  • Binds viral glycoproteins 


Narcissus Pseudonarcissus Lectin (NPA, NPL) 

  • Preferential binding towards α1,6-linked mannose 
  • Does not bind glucose 
  • Binds galactomannans, polysaccharides containing a mannose backbone, and galactose side groups 


Complex N-glycan Binding Lectins 

The following lectins predominantly recognize complex N-glycans, specifically biantennary N-glycans. 

Phaseolus Vulgaris-L (PHA-L) 

  • Specific towards β1,6-branched N-glycans 
  • Binds tri- and tetraantennary structures 
  • Specificity is reduced by α2,6 sialylation, but not α2,3 sialylation, core fucosylation, or bisecting GlcNAc 


Core O-glycan Binding Lectins 

O-glycan epitopes form as a result of the glycosylation of a serine or threonine residue by Nacetylgalactosamine (GalNAc). These epitopes, also called Tn antigens, are abundant in mucin cores and glycopeptides found on epithelial cells of several tissues in the body, and are overexpressed in cancer cells in breast, pancreas, prostate, and lung epithelium (9). The following lectins have high specificity towards O-glycan epitopes: 

Artocarpus Integrifolia (AIA, Jacalin) 

  • Predominantly binds 3-substituted GalNAcα epitopes, called core 1 and core 3 O-glycans 
  • Tolerated by α- or β- linkages of oligosaccharides, including GalNAc, GlcNAc, and Gal 
  • Inhibited by substitution at the 6 position  


Fucose Binding Lectins 

Fucosylation is a terminal modification via α1,6 linkage, occurring on asparagine-linked GlcNAc of hybrid and complex N-glycans. In particular, α1,3- and α1,4-fucosylation is associated with cancer metastasis (10). 

Aleuria Aurantia Lectin (AAL) 

  • Preferential binding towards Fucα1,2-terminated structures on type 2 LacNAc 
  • Preferential binding to fucose linked (α1,6) to N-acetylglucosamine or fucose linked (α1,3) to N-acetyllactosamine 


Sialic Acid and Sulfate Binding Lectins 

Sialylation involves the binding of sialic acid to N– and O-glycans via various glycosidic linkages, such as α2,3-, α2,6-, and α2,8-, while sulfation is more common to glycosaminoglycans. 

Maackia Amurensis-II (MAL-II) 

  • Recognizes sialic acid on O-glycans 
  • Preferential binding to α2,3-sialylated Galβ1−3GalNAc in O-glycans 
  • Binding tolerates substitutions at the 6-position of GalNAc 
  • 3’O-sulfated Gal epitope is the main binding motif 
  • Disulfation and fucosylation on this motif inhibit binding 


Terminal Gal and LacNAc Binding Lectins 

Terminal Gal and LacNAc residues have significant roles in glycoconjugate function. Terminal glycan modifications, such as sialylation and fucosylation, occur on these residues. Improper structural changes on these terminal residues are key biomarkers of disease. 

Ertythrina Cristagalli Agglutinin (ECL, ECA) 

  • Specific recognition of terminal Type 2 LacNAc 
  • Sialic acid substitution inhibits binding 
  • Useful in isolating human-natural killer (NK) cells 


Wisteria Floribunda Agglutinin (WFA) 

  • Recognizes terminal GalNAc structures bearing LacdiNAc 
  • The primary binding motif is β-GalNAc, including β-GalNAc terminated glycosphingolipid structures 
  • Recognizes terminal α-GalNAc of simple 1-2 sugars 
  • Tolerates substitution on the proximal residue 


Terminal GlcNAc and Chitin Binding Lectins 

Wheat Germ Agglutinin (WGA) 

  • A widely studied lectin with broad specificity for various glycans containing GlcNAc 
  • The principal binding motif is terminal GlcNAcβ 
  • Preferential binding to long-chain polyLacNAc and multiantennary N-glycans 
  • Other binding residues include GlcNAcα-, GalNAcα-, GalNAcβ-, and MurNAcβ- 


Resources to help you​ 

Lectins are helpful tools for analyzing the relationship between glycan composition and cellular function in both healthy and abhorrent samples. To fully appreciate the potential lectins will have in modern medicine, we need to expand our knowledge of their binding mechanisms and specificity. 

If you plan to incorporate lectins in your glycan research, we recommend looking at “A Useful Guide to Lectin Binding: Machine-Learning Directed Annotation of 57 Unique Lectin Specificities” from the Mahal Lab at the University of Alberta as well as the National Center for Functional Glycomics (NCFG). 


There are numerous ways to identify and analyze cellular glycans with lectins, so it may be challenging to determine the most appropriate method for your specific needs. At Vector Laboratories, our aim is to inform and support you with detailed explanations and demonstrations of lectin workflows. To learn more about lectin applications, you can visit our Lectins resources page, where you can find tools that will help you incorporate these powerful molecules into your workflow. If you’re interested in the specific lectins discussed in this article, check out our Glycan Screening Kits for an easy, streamlined approach to detect glycan expression in your tissues.  


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  2. Weis WI, et al. 1996. Structural Basis of Lectin-Carbohydrate Recognition. Annual Review of Biochemistry. 
  3. Hester G, et al. 1995. Structure of Mannose-Specific Snowdrop (Galanthus nivalis) Lectin is Representative of a New Plant Lectin Family. Nature Structural & Molecular Biology 
  4. Kaushik S, et al. 2009. The Role of Metal Ions in Substrate Recognition and Stability of Concanavalin A: A Molecular Dynamics Study. Biophysical Journal. 
  5. Mishra A, et al. 2019. Structure-Function and Application of Plant Lectins in Disease Biology and Immunity. Food and Chemical Toxicology. 
  6. Kletter D, et al. 2018. Determining Lectin Specificity From Glycan Array Data Using Motif Segregation and GlycoSearch Software. Current Protocols in Chemical Biology. 
  7. Bojar D, et al. 2022. A Useful Guide to Lectin Binding: Machine-Learning Directed Annotation of 57 Unique Lectin Specificities. ACS Chemical Biology. 
  8. Lavine CL, et al. 2012. High-Mannose Glycan-Dependent Epitopes Are Frequently Targeted in Broad Neutralizing Antibody Responses During Human Immunodeficiency Virus Type 1 Infection. Journal of Virology. 
  9. Kufe DW. 2009. Mucins in Cancer: Function, Prognosis and Therapy. Nature Reviews Cancer. 
  10. Agrawal P, et al. 2017. A Systems Biology Approach Identifies FUT8 as a Driver of Melanoma Metastasis. Cancer Cell.