Z Biotech’s Neu5Ac/Neu5Gc N-glycan microarray helps researchers track changes in host glycan targets of avian influenza within H5N1 phylogenies.

From Birds to Mammals: The Role of Glycan Specificity in H5N1 Transmission

Highly pathogenic avian influenza H5N1 continues to raise global public health concerns due to its zoonotic potential. Recent outbreaks have shown that H5N1 can infect dairy cows. This unusual host range is partly explained by glycan receptor specificity: avian influenza viruses predominantly bind glycans terminating in α2-3 linked sialic acids, while human influenza viruses bind glycans terminating in α2-6 linked sialic acids. Dairy cow mammary tissues, the primary site of H5N1 infection, uniquely express both α2-3 and α2-6 linked sialic acids, positioning dairy cows as a potential bridge for H5N1 transmission between avian species and humans.

Understanding the molecular mechanisms that enable such host-range expansions is crucial for surveillance and pandemic preparedness. Two recent studies provide critical insights into how single mutations in H5N1 hemagglutinin (HA) influence glycan binding specificity and host adaptation.

Mutations in H5N1 HA and Their Impacts on Glycan Binding

Increased Binding Breadth through a Single Mutation

The study led by Jenna J. Guthmiller from the University of Colorado Anschutz Medical Campus identified that a single mutation (T199I) in the HA of dairy cow-associated H5N1 (A/Texas/37/2024) increased receptor binding breadth to glycans terminating in α2-3 sialic acids. Structural analyses revealed that this mutation enhances the flexibility of the receptor-binding site (RBS), enabling broader glycan recognition. While this mutation did not confer affinity for α2-6 sialic acids, it highlights how small genetic changes can significantly impact host range and interspecies transmission potential​.

 

 

Figure 1. The glycan binding profiles of WT A/Texas/37/2024 and its T199I mutant were determined using a glycan array, revealing that the T199I mutation is responsible for the increased glycan binding breadth in A/Texas/37/2024.
The figure is reproduced from Guthmiller, J. J., et al. A single mutation in dairy cow-associated H5N1 viruses increases receptor binding breadth. Nature Communications, 15, 1234 (2024).

Switching Receptor Specificity to Human-Type Glycans

The study led by Ian A. Wilson and James C. Paulson from The Scripps Research Institute demonstrated that a single Gln226Leu substitution in the same H5N1 strain completely switched receptor specificity from α2-3 to α2-6 linked sialic acids, characteristic of human influenza viruses. This mutation, when combined with Asn224Lys, further enhanced human-type receptor binding. Structural studies provided mechanistic insights, showing how these mutations reorient critical interactions within the RBS to favor human glycans. These findings underscore the potential for H5N1 to adapt for human-to-human transmission, emphasizing the need for vigilant monitoring of emerging mutations.

 

 

Figure 2. The binding specificity of the Gln226Leu mutation was determined using a glycan array, revealing that the mutation switches specificity entirely to α2-6 sialosides while eliminating binding to α2-3 sialosides. This finding underscores the critical role of specific mutations in altering HA receptor preferences and provides key insights into the potential adaptation of H5N1 for human infection.

The figure is reproduced from Lin, T.-H., Zhu, X., Wang, S., Zhang, D., McBride, R., Yu, W., Babarinde, S., Paulson, J. C., & Wilson, I. A. A single mutation in bovine influenza H5N1 hemagglutinin switches specificity to human receptors. Science, 386, 1128–1134 (2024)

The Role of Glycan Arrays in Influenza Research

Both studies relied heavily on glycan array technology to dissect the glycan-binding properties of H5N1 HA. Glycan arrays enabled researchers to rapidly and comprehensively profile HA interactions with a diverse set of glycans, revealing subtle differences in binding preferences that are not easily detectable through traditional assays. These platforms provided:

• Quantitative binding data for α2-3 and α2-6 sialic acids.
• Comparative analysis of wild-type and mutant HAs, highlighting the impact of specific mutations.
• Structural insights by correlating binding data with cryo-EM and crystallographic studies.

The ability of glycan arrays to resolve these complex interactions has made them indispensable tools for understanding viral glycan specificity and guiding public health strategies.

The Value of the Neu5Gc/Neu5Ac N-Glycan Array

Our Neu5Gc/Neu5Ac N-Glycan array represents an advanced platform for investigating glycan-binding interactions in various biological contexts, including influenza HA studies. With 44 structurally defined N-glycans terminating in either Neu5Gc or Neu5Ac, this array provides unparalleled resolution for exploring sialoglycan interactions. Key advantages include:

• High-throughput capacity with 8 or 16 subarrays for simultaneous sample analysis.
• Minimal sample volume requirements and rapid assay turnaround.
• Customizable design to meet specific research needs.

For researchers studying influenza or other glycan-mediated biological processes, the Neu5Gc/Neu5Ac N-Glycan array offers a powerful and efficient solution. By facilitating the identification of glycan-binding preferences, this array can accelerate discoveries in virology, immunology, and therapeutic development.

Conclusion

The two studies reviewed here underscore the critical role of glycan specificity in shaping the host range and zoonotic potential of H5N1. Glycan array technology was pivotal in uncovering these molecular insights, demonstrating the transformative impact of these tools in glycoscience. With the Neu5Gc/Neu5Ac N-Glycan array, researchers have access to a robust platform for advancing our understanding of sialoglycan interactions, offering new avenues for influenza research and beyond.