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.

Keywords: Calorimetry; Glycosylation; Lectin; Proliferation; Protein design; p53.

Fig. 1

Modular architecture of the tested galectins: examples of the three types of vertebrate galectins (top row), the two homodimeric variants of Gal-7 established by direct or linker-mediated connection (middle) and the panel of heterodimers with directly conjugated CRDs and CRDs connected by the 33-amino-acid linker of Gal-8S (bottom)

Fig. 2

Illustration of gel electrophoretic analysis of Gal-7 (control) along with its homodimeric variants (a) or with its heterodimeric variants containing the CRD of Gal-1 (b) or of Gal-3 (c). Positions of marker proteins for mass calibration and quantity of protein are given

Fig. 3

Mass spectrum of Gal-7–8S–Gal-7 (a), peptide mass fingerprint (b) and illustration of sequence coverage by fingerprinting that reaches a value of 94.1% (c). Sequence of the inserted 33-amino-acid linker of Gal-8S is highlighted in green

Fig. 4

Thermograms of LacNAc (6 mM) association to human Gal-7 (a) and to the heterodimers Gal-1–Gal-7 (b), Gal-1–8S–Gal-7 (c) and Gal-3–8S–Gal-7 (d)

Fig. 5

Illustration of the inhibitory effect of cognate sugar (Lac) on staining profiles with the biotinylated Gal-7–Gal-7 homodimer in cross sections through the fixed murine epididymis (initial segment; a–d) or jejunum (e–h). Strong staining intensity in the epididymis (a) was reduced in stepwise fashion by the presence of 0.2 mM or 5 mM Lac (b, c). Bivalent glycocluster (at 0.2 mM Lac) was more effective than tetravalent compound (d, inset to d). Galectin binding in the jejunum was blocked by Lac present in the bivalent glycocluster (control: e; sugar concentrations of 0.1 mM, 0.5 mM and 1 mM: f–h), insets to f–h showing respective activity of free Lac at 0.1 mM, 0.5 mM and 20 mM. The galectin was applied at 4 µg/mL. Scale bars are 20 µm

Fig. 6

Illustration of staining profiles by labeled Gal-7 and Gal-7-based variants in cross sections of fixed murine epididymis (initial segment a–f) and jejunum (g–l). Extent of positivity appeared rather similar in principal (arrow), apical (white arrowhead) and basal (black arrowhead) cells with Gal-7 (a), its directly conjugated homodimer (b) and the Gal-7–Gal-1 heterodimer (c). Insertion of the peptide linker led to intensity reduction, strongly in this case and less so in the Gal-1–Gal-7 protein (d and inset; arrowhead: basal cell). The marked impact of linker presence on the signal intensity of basal cells (arrowhead) is documented in the case of the Gal-7–Gal-3 heterodimer pair (e, f). Murine jejunum presented similar staining profiles for Gal-7 (g), its directly linked homodimer (h) and the Gal-7–Gal-1 heterodimer (i); surface enterocytes (black arrowhead) and contents of goblet cells (white arrowhead), crypt cells (arrow) and lamina propria cells (asterisks) are highlighted. Presence of linker (inset to h) and change in CRD position in the dimer (inset to i) have minor effects. Presence of linker showed a strong reduction for the Gal-7–Gal-1 heterodimer (j), less so for its sequence permutation-based protein (inset). The same impact is observed for the Gal-7–Gal-3 heterodimer pair (k, l; highlighting by symbols as in g). Scale bars are 20 µm

Fig. 7

At-a-glance illustration of differences in staining patterns of labeled Gal-7 and the heterodimer pair with sequence permutation, i.e. Gal-7–Gal-3 and Gal-3–Gal-7 in villi (a) and the base of crypts (b) in sections of fixed murine jejunum. (a) Brush border (asterisk), surface enterocytes (white arrowhead), immune cells of the lamina propria (black arrow) and contents of goblet cells (black arrowhead) are highlighted to emphasize distinct aspects, as done in (b) for the prominent difference in staining intensity of cells at the base of crypts (arrow; for summary of intensity of staining, please see Table 3). Scale bars are 10 µm

Fig. 8

Inhibition of neuroblastoma cell proliferation by wild-type galectins and the panel of engineered variants (**p < 0.01; ***p < 0.001)