Human chimera-type galectin-3: Defining the critical tail length for high-affinity glycoprotein/cell surface binding and functional competition with galectin-1 in neuroblastoma cell growth regulation

• Trimodular galectin-3 is redesigned for activity assays.
• In-source decay mass spectrometry ascertains quality of variants.
• A switch-like site for regulating cis-binding is identified.
• Activity for cell bridging is maintained despite drastic tail truncation.

Many human proteins have a modular design with receptor and structural domains. Using adhesion/growth-regulatory galectin-3 as model, we describe an interdisciplinary strategy to define the functional significance of its tail established by nine non-triple helical collagen-like repeats (I–IX) and the N-terminal peptide. Genetic engineering with sophisticated mass spectrometric product analysis provided the tools for biotesting, i.e. eight protein variants with different degrees of tail truncation. Evidently,various aspects of galectin-3 activity (cis binding and cell bridging) are affected by tail shortening in a different manner. Thus, this combined approach reveals an unsuspected complexity of structure–function relationship, encouraging further application beyond this chimera-type galectin.

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