Tissue lectins are emerging (patho)physiological effectors with broad significance. The capacity of adhesion/growth-regulatory galectins to form functional complexes with distinct cellular glycoconjugates is based on molecular selection of matching partners. Engineering of variants by changing the topological display of carbohydrate recognition domains (CRDs) provides tools to understand the inherent specificity of the functional pairing. We here illustrate its practical implementation in the case of human tandem-repeat-type galectin-8 (Gal-8). It is termed Gal-8 (NC) due to presence of two different CRDs at the N- and C-terminal positions. Gal-8N exhibits exceptionally high affinity for 3′-sialylated/sulfated β-galactosides. This protein is turned into a new homodimer, i.e., Gal-8 (NN), by engineering. The product maintained activity for lactose-inhibitable binding of glycans and glycoproteins. Preferential association with 3′-sialylated/sulfated (and 6-sulfated) β-galactosides was seen by glycan-array analysis when compared to the wild-type protein, which also strongly bound to ABH-type epitopes. Agglutination of erythrocytes documented functional bivalency. This result substantiates the potential for comparative functional studies between the variant and natural Gal-8 (NC)/Gal-8N.

Galectin-1 (Gal-1) is a galactose-binding protein involved in various cellular functions. Gal-1’s activity has been suggested to be connected to two molecular concepts, which are, however, lacking experimental proof: a) enhanced binding affinity of Gal-1 toward membranes containing monosialotetrahexosylganglioside (GM₁) over disialoganglioside GD₁a and b) cross-linking of GM₁’s by homodimers of Gal-1. We provide evidence about the specificity and the nature of the interaction of Gal-1 with model membranes containing GM₁ or GD₁a, employing a broad panel of fluorescence-based and label-free experimental techniques, complemented by atomistic biomolecular simulations. Our study demonstrates that Gal-1 indeed binds specifically to GM₁ and not to GD₁a when embedded in membranes over a wide range of concentrations (i.e., 30 nM to 20 μM). The apparent binding constant is about tens of micromoles. On the other hand, no evidence of Gal-1/GM₁ cross-linking was observed. Our findings suggest that cross-linking does not result from sole interactions between GM₁ and Gal-1, indicating that in a physiological context, additional triggers are needed, which shift the GM₁/Gal-1 equilibria toward the membrane-bound homodimeric Gal-1.

Extracellular vesicles (EVs) are emerging as diagnostic and therapeutic agents, yet their nanoscale size limits quantitative single‐vesicle analysis of surface epitopes and molecular interactions. Flow‐Induced Dispersion Analysis (FIDA) is a microfluidic technique enabling real‐time measurement of hydrodynamic size and interaction kinetics using fluorescent ligands without requiring surface immobilization. Here, FIDA was applied to characterize EV–antibody interactions using anti‐CD63 antibodies across three EV sources: EVs from genetically engineered HEK cells expressing CD63‐eGFP or CD63‐NeonGreen‐Fc domain (CD63‐NG‐Fc), and EVs from clonally expanded immortalized mesenchymal stromal cells (ciMSCs). FIDA‐measured EV diameter sizes ranged from 40 to 90 nm, compared to 70–200 nm as determined by Nanoparticle Tracking Analysis (NTA), likely reflecting methodological differences or NTA's sensitivity to non‐EV particles. CD63‐binding EC ₅₀  values were 1.9 × 10 ⁸ particles/mL for CD63‐eGFP EVs and 8.4 × 10 ⁸ to 2.4 × 10 ⁹ particles/mL for ciMSC EVs, indicating higher epitope abundance in engineered vesicles. Within a broader antibody titration, maximal antibody loading to the Fc‐receptor domain on EVs was observed at 0.5–5 nM, reaching 30%–40% for IgG1 and 15%–20% for Cetuximab. These results establish FIDA as a high‐resolution, label‐efficient tool for quantifying EV‐antibody interactions and epitope accessibility, supporting its integration into EV standardization and quality control workflows.

Galectin-1 (Gal-1) is a galactose-binding protein involved in various cellular functions. Gal-1’s activity has been suggested to be connected to two molecular concepts, which are however lacking experimental proof: a) enhanced binding affinity of Gal-1 towards membranes containing monosialotetrahexosylganglioside (GM ₁ ) over disialoganglioside GD ₁ a and b) cross-linking of GM ₁ ’s by homodimers of Gal-1. We provide evidence about the specificity and the nature of Gal-1 interaction with model membranes containing GM ₁ or GD ₁ a, employing a broad panel of fluorescence-based and label-free experimental techniques, complemented by atomistic biomolecular simulations. Our study demonstrates that Gal-1 binds indeed specifically to GM ₁ , and not to GD ₁ a, when embedded in membranes over a wide range of concentrations (i.e., 30 nM to 10 μM). The apparent binding constant is about tens of micromoles. On the other hand, no evidence of Gal-1/GM ₁ cross-linking was observed. Our findings suggest that cross-linking does not result from sole interactions between GM ₁ and Gal-1, indicating that in a physiological context, additional triggers are needed, which shift the GM ₁ /Gal-1 equilibria towards the membrane-bound homodimeric Gal-1. Abstract Figure: