Glycans form intricate, dense envelopes around cells, modulating interactions in the biological environment. Unlike DNA or proteins, glycans are profoundly diverse in structure, making them notoriously difficult to analyze and characterize. Standard investigative tools have fallen short in targeting these sugar moieties, mainly because glycans do not elicit strong immune responses or display easily recognizable sequences. The challenge has long been to develop a molecular “spotlight” capable of selectively binding and illuminating specific glycan structures, thereby revealing their functions in health and disease.

The University at Buffalo team focused their efforts on ST3Gal1, a glycosyltransferase derived from pigs, which naturally catalyzes the addition of sialic acid residues to forming glycan chains. By introducing a strategically chosen mutation, termed H302A, they effectively abolished the enzyme’s catalytic activity—disabling its sugar-synthesizing capabilities—while simultaneously converting it into a binding agent with high affinity for sialylated core-2 O-glycans. This reimagined enzyme, suitably named sCore2, acts as a molecular probe that can latch onto these sugar residues without altering them, a feat that opens broad horizons in glycan detection.

Developing sCore2 demanded not only the mutation of ST3Gal1 but also sophisticated enhancement techniques. Employing mammalian surface-display technology, the team expressed the mutant enzyme on the exterior of mammalian cells, harnessing the natural cellular machinery to foster proper folding and display of functional proteins. This approach optimized sCore2’s ability to interact with its target glycans under physiologically relevant conditions, a critical step that improves the probe’s specificity and binding strength in complex biological environments.

To visualize sCore2’s binding events, the researchers conjugated the enzyme to fluorescent antibodies. This engineering allowed the probe to emit a detectable glow upon binding to sialylated core-2 O-glycans, facilitating visualization through advanced imaging techniques like flow cytometry and fluorescence microscopy. Testing on human blood and tissue samples corroborated the presence of these glycans predominantly on mature immune cells and within certain cancerous tissues, notably breast cancer. Moreover, sCore2 unveiled glycan presentations not previously observed in organs such as the spleen and pancreas, hinting at unexplored biological functions.

The implications of these findings extend deeply into disease biology, as the differential expression of sialylated core-2 O-glycans could serve as reliable biomarkers for cancer detection and immune cell phenotyping. The ability to pinpoint these sugars provides a new dimension in diagnosing pathological states and monitoring immune responses. Additionally, the tool sets a precedent for the design of a broader repertoire of custom glycan-binding proteins, potentially culminating in a comprehensive “dictionary” of sugar-recognizing molecules tailored to various biomedical applications.

Beyond the immediate scope of glycobiology, this research signals a paradigm shift in enzyme engineering. The concept of reversing enzyme functionality—transforming synthetic glycosyltransferases into binding proteins—illustrates a versatile platform approach. As noted by Dr. Sriram Neelamegham, the corresponding author and Distinguished Professor at UB, this strategy could be extrapolated to human genes and diverse enzyme classes, enabling scientists to construct sophisticated molecular sensors for many elusive biomolecules.

This advancement also demonstrates the significance of structural biology in guiding molecular design. The team employed detailed knowledge of the enzyme’s three-dimensional conformation and binding interfaces to rationally select the H302A mutation, ensuring the switch in enzymatic behavior without compromising structural integrity. The availability of high-resolution structural models facilitated targeted alterations and propagation of desirable binding properties.

The ongoing development of sCore2 and related probes is fuelled by interdisciplinary collaboration among the university’s departments of Biomedical Engineering, Pathology and Anatomical Sciences, and Medicine. Their collective expertise spans enzymology, chemical biology, and biophysics, culminating in this innovative glycan-detection system. This convergence underscores the increasing importance of cross-disciplinary approaches in solving complex biological puzzles.

Protecting the intellectual property associated with sCore2, the researchers have filed a provisional patent, safeguarding both the composition of the engineered enzyme and the methodologies underpinning its creation. Funding from the National Institutes of Health and the UB Center of Excellence in Bioinformatics and Life Sciences continues to propel this promising avenue of research forward, with the team pursuing further refinements and applications.

In sum, this pioneering work heralds a new era in the exploration of cellular sugar landscapes. By equipping scientists with a versatile, highly specific glycan-binding probe, the study illuminates previously hidden dimensions of cell biology and disease. As the toolkit expands, so too will our capacity to diagnose, monitor, and potentially intervene in diseases through the language of cellular carbohydrates, a realm once deemed too complex to decode.

Subject of Research: Engineering glycosyltransferases into glycan-binding proteins to enable specific detection of sialylated core-2 O-glycans on cells.

Article Title: Engineering glycosyltransferases into glycan binding proteins using a mammalian surface display platform

News Publication Date: August 14, 2025

Web References:

• https://www.nature.com/articles/s41467-025-62018-z#author-information
• http://dx.doi.org/10.1038/s41467-025-62018-z

References:

• Previous related study in Small: https://onlinelibrary.wiley.com/doi/full/10.1002/smll.202502318
• Review article in Glycobiology: https://academic.oup.com/glycob/advance-article/doi/10.1093/glycob/cwaf041/8210292

Image Credits: University at Buffalo

Keywords

Cell biology, Life sciences, Chemical biology, Structural biology, Protein functions, Pharmacology, Glycobiology, Enzymology, Biochemical processes, Molecular targets

Central to this innovative vaccine’s success is its ability to precisely incorporate two pivotal viral proteins: hemagglutinin (H5) and neuraminidase (N1). These proteins are integral to the virus’s infectious cycle, with hemagglutinin facilitating viral entry into host cells, while neuraminidase plays a crucial role in the release and spread of new viral particles. Unlike many existing vaccines that primarily target the hemagglutinin protein, this platform explores a bivalent approach, combining immune targets to potentially enhance protection and broaden the vaccine’s efficacy against viral mutations.

The platform leverages recombinant protein technology, eschewing traditional egg-based vaccine production methods. Instead, the H5 and N1 proteins are engineered with a histidine tag—a short amino acid sequence with a natural affinity for metals—that allows them to bind efficiently and specifically to cobalt ions embedded within cobalt-porphyrin-phospholipid (CoPoP) nanoparticles. This nanoparticle scaffold forms the core of the vaccine delivery system, providing a stable and versatile platform that presents antigens in a manner that effectively stimulates the immune system.

Preclinical trials conducted on mice exhibited compelling results: administration of hemagglutinin alone conferred full protection, completely preventing signs of illness, weight reduction, and viral replication within lung tissues. The neuraminidase-only formulation, while providing partial immunity with approximately 70% effectiveness, demonstrated the capacity to reduce viral load and disease severity, underscoring the importance of neuraminidase antibodies in modulating infection. Interestingly, the combination of H5 and N1 as a bivalent vaccine did not surpass the efficacy observed with hemagglutinin alone, suggesting a predominant role for hemagglutinin in protective immunity but reaffirming the supportive benefits of neuraminidase-targeted responses.

The CoPoP nanoparticle’s design not only supports antigen presentation but also incorporates potent adjuvants—including QS-21, a saponin derivative known to enhance cellular and humoral immune responses, and PHAD, a synthetic monophosphoryl lipid A derivative acting as a Toll-like receptor 4 agonist. Both adjuvants are embedded within the phospholipid bilayer shell, amplifying the vaccine’s immunogenicity by promoting a robust and durable immune activation. This molecular synergy enables the platform to elicit broad-spectrum protection with potentially improved durability and response quality compared to conventional vaccines.

What distinctly sets this vaccine platform apart is its manufacturing advantage. Traditional influenza vaccines rely heavily on egg-based propagation of live or attenuated viruses—a time-consuming process susceptible to supply chain constraints. In contrast, the UB strategy produces antigenic proteins through recombinant expression systems, which are then effortlessly conjugated to nanoparticles via rapid and stable metal-affinity interactions. This method promises expedited vaccine production timelines, scalability, and adaptability critical in responding swiftly to emergent virus strains during pandemics or zoonotic spillovers.

The CoPoP nanoparticle technology underlying this vaccine platform is not a nascent concept; it has undergone advanced clinical evaluations in unrelated viral contexts, notably as a COVID-19 vaccine candidate. These phase 2 and 3 trials, conducted in collaboration with industry partners and international research bodies, have demonstrated the platform’s safety and immunogenic profile in humans, bolstering confidence that the technology can be effectively translated into licensed vaccines for other pathogens, including avian influenza.

From a molecular perspective, the strategic use of histidine-tagged antigens exploits the affinity between imidazole side chains of histidine residues and transition metal ions, fostering swift and stable antigen attachment without compromising protein conformation or function. This design ensures that the antigens display native epitopes essential for inducing neutralizing antibodies and T-cell responses, a feat difficult to achieve in many subunit vaccine approaches.

Moreover, the research highlights the nuanced roles of viral glycoproteins in immune defense. Hemagglutinin serves as the viral key for host cell interaction, dictating entry specificity and initial infection, which makes it a prime neutralizing antibody target. Neuraminidase, acting as an enzymatic scissors, cleaves sialic acid residues to facilitate virion release, and while antibodies targeting N1 are non-neutralizing in the classical sense, they reduce viral dissemination and disease severity, contributing to overall vaccine efficacy. This understanding of immunological mechanisms reinforces the rationale for including multiple antigenic components to counteract viral escape mutations.

Looking ahead, the UB team intends to expand their evaluations by experimenting with dosage variations, vaccination schedules, and administration routes to optimize the vaccine’s protective effect and practical deployment. The multi-institution collaboration, spanning public health agencies, national microbiology laboratories, veterinary research centers, and biotech firms, exemplifies the integrative approach necessary to combat complex zoonotic threats effectively.

The promise of this vaccine platform extends beyond avian influenza. Its modular design, speed of production, and potent immune activation could serve as a blueprint for rapid response vaccines against other emerging infectious diseases. In an era marked by the continuous emergence of viral variants with pandemic potential, innovative technologies such as this herald a new paradigm in vaccinology, where precision engineering, nanotechnology, and immunology converge to safeguard both animal and human health.

The research, slated for publication in the prestigious journal Cell Biomaterials, epitomizes cutting-edge advances that could redefine influenza vaccination frameworks and fortify global preparedness against evolving viral pathogens.

Subject of Research: Avian influenza vaccine development targeting H5N1 variant 2.3.4.4b using a cobalt-porphyrin-phospholipid nanoparticle platform.

Article Title: University at Buffalo Develops Novel Nanoparticle Platform Achieving Complete Protection Against Deadly H5N1 Avian Influenza Variant in Mice

News Publication Date: 17-Apr-2025

Image Credits: University at Buffalo

Keywords: Avian influenza, Flu vaccines, Animal research, Influenza viruses, Bond formation, Vaccine development, Wild birds, Public health, COVID 19, Recombinant proteins, Cell division, Animal models