Imitating evolution’s tinkering by protein engineering reveals extension of human galectin-7 activity

Abstract

Wild-type lectins have distinct types of modular design. As a step to explain the physiological importance of their special status, hypothesis-driven protein engineering is used to generate variants. Concerning adhesion/growth-regulatory galectins, non-covalently associated homodimers are commonly encountered in vertebrates. The homodimeric galectin-7 (Gal-7) is a multifunctional context-dependent modulator. Since the possibility of conversion from the homodimer to hybrids with other galectin domains, i.e. from Gal-1 and Gal-3, has recently been discovered, we designed Gal-7-based constructs, i.e. stable (covalently linked) homo- and heterodimers. They were produced and purified by affinity chromatography, and the sugar-binding activity of each lectin unit proven by calorimetry. Inspection of profiles of binding of labeled galectins to an array-like platform with various cell types, i.e. sections of murine epididymis and jejunum, and impact on neuroblastoma cell proliferation revealed no major difference between natural and artificial (stable) homodimers. When analyzing heterodimers, acquisition of altered properties was seen. Remarkably, binding properties and activity as effector can depend on the order of arrangement of lectin domains (from N- to C-termini) and on the linker length. After dissociation of the homodimer, the Gal-7 domain can build new functionally active hybrids with other partners. This study provides a clear direction for research on defining the full range of Gal-7 functionality and offers the perspective of testing applications for engineered heterodimers.

In focus in HCB

“…The Gabius research group has been leading such investigations for many years (for instance, Roy et al 2017). In this current issue, they provide a very detailed and well-presented example of using protein engineering to investigate the intriguing question of why evolutionary mechanisms resulted in a specific lectin CRD, rather than any of the multitudinous variations also possible (Ludwig et al 2021). As a test lectin, they chose to use galectin-7 (Gal-7), an N-acetyllactosamine-specific member of the vertebrate galectin family, whose CRD may be arranged in three types of structures: a non-covalently associated homodimer, a linker-coupled heterodimer, or a CRD combined with a structurally different second part.…”

“…Fig.3Illustration of differences in staining patterns of labeled Gal-7 and the heterodimer pair with sequence permutation (Gal-7-Gal-3 and Gal-3-Gal-7) in villi in sections of murine jejunum. FromLudwig et al (2021) …”

Perturbing dimer interactions and allosteric communication modulates the immunosuppressive activity of human galectin-7

“…, GAL-4 ( 20 , 21 ). To further complicate matters, an increasing number of studies have now confirmed the importance of GBS-independent activities modulated by galectins ( 22 , 23 , 24 ), including potentially relevant hetero-oligomeric galectin architectures, modular designs, and valence variability ( 25 , 26 , 27 , 28 ). This should come as no surprise, as it has been known for a while that lectins can bind noncarbohydrate compounds, often exhibiting higher affinities than their “natural” saccharide ligands ( 29 ).…”

In Memoriam

“…Hans-Joachim Gabius realised the aptitude of fixed tissue sections with cellular glycomes of normal and pathological origin as an in vivo assay platform to study molecular interactions between naturally occurring or engineered (ga) lectins and their respective ligands. This system could be further expanded to test the inhibitory capacity of potential therapeutic glycocompounds on these interactions (Ludwig et al 2021).…”