A Synthetic Tetramer of Galectin-1 and Galectin-3 Amplifies Pro-apoptotic Signaling by Integrating the Activity of Both Galectins

Abstract

Galectin-1 (G1) and galectin-3 (G3) are carbohydrate-binding proteins that can signal apoptosis in T cells. We recently reported that a synthetic tetramer with two G1 and two G3 domains ("G1/G3 Zipper") induces Jurkat T cell death more potently than G1. The pro-apoptotic signaling pathway of G1/G3 Zipper was not elucidated, but we hypothesized based on prior work that the G1 domains acted as the signaling units, while the G3 domains served as anchors that increase glycan-binding affinity. To test this, here we studied the involvement of different cell membrane glycoproteins and intracellular mediators in pro-apoptotic signaling via G1/G3 Zipper, G1, and G3. G1/G3 Zipper induced Jurkat T cell death more potently than G1 and G3 alone or in combination. G1/G3 Zipper, G1, and G3 increased caspase-8 activity, yet only G1 and G3 depended on it to induce cell death. G3 increased caspase-3 activity more than G1/G3 Zipper and G1, while all three galectin variants required it to induce cell death. JNK activation had similar roles downstream of G1/G3 Zipper, G1, and G3, whereas ERK had differing roles. CD45 was essential for G1 activity, and was involved in signaling via G1/G3 Zipper and G3. CD7 inhibited G1/G3 Zipper activity at low galectin concentrations but not at high galectin concentrations. In contrast, CD7 was necessary for G1 and G3 signaling at low galectin concentration but antagonistic at high galectin concentrations. Collectively, these observations suggest that G1/G3 Zipper amplifies pro-apoptotic signaling through the integrated activity of both the G1 and G3 domains.

Physical tuning of galectin-3 signaling

“…It is well known that galectin-1 is active as a homodimer, which supports the observed function of the heterotetramer, whereas the minimal functional oligomer of Gal3 was unknown. Here, we showed that the minimum valency for Gal3 activity is also 2, consistent with a report showing that both galectin-1 and Gal3 contribute to the signaling activity of the heterotetramer (37). In contrast to the heterotetramer design, here the Gal3 domains were fused to one terminus of a parallel, α-helical coiled-coil while sfGFP was fused to the other.…”

“…It is valuable to understand the biological mechanisms and outcomes of these promiscuous Gal3receptor interactions for pharmaceutical applications or drug discovery (e.g., design of selective Gal3 inhibitors or biomimetic Gal3 agonists). In addition to the Jurkat T cell line, two different receptor-deficient lymphocyte cell lines, J45.01 (CD45 − /CD7 + ) and HuT 78 (CD45 + /CD7 − ), have been used previously to characterize the role of CD45 and CD7 in wild-type Gal3-mediated membrane glycan clustering and cell death signaling (36,37). Unlike J45.01 and HuT 78 T cells, Jurkat T cells express both CD45 and CD7, which differ both in size and availability on the cell surface (Fig.…”

Altering the Modular Architecture of Galectins Affects its Binding with Synthetic α‐Dystroglycan O‐Mannosylated Core M1 Glycoconjugates In situ

“…Presenting Gal-1 into a tandem-repeat type with an 8S-linker ((Gal-1) 2 [8S]) maintained its crosslinking activity with laminin and α-DG peptide 4, but attenuated transbridging with core M1 glycopeptides (5--11) was observed. Interestingly, a noticeable increase in trans-bridging towards LacNAc-terminated (12)(13)(14)(15)(16)(17)(18)26) and sialyl-LacNAc-terminated (19)(20)(21)(22)(23)(24)(25)(27)(28)(29) was demonstrated by (Gal-1) 2 [8S] which is attributed to the high affinity of this Gal-1 variant to the ligands compared to Gal-1WT (Figure 6). This difference in crosslinking activity is associated with the Gal-8 linker, 33 aa (8S), between the two Gal-1 monomers providing Gal-8like properties to Gal-1.…”

“…[12] Furthermore, a heterotetramer galectin variant with two Gal-1 and Gal-3 domains (G1/G3 Zipper) exhibited a higher apoptotic activity than wild-type Gal-1 and Gal-3 alone or in combination; thus, it can be a good therapeutic candidate for regulating innate and adaptive immunity. [20,21] Dystroglycan (DG) is a transmembrane protein that links the extracellular matrix and intracellular cytoskeleton via α-DG and β-DG, respectively. [22] α-DG is highly O-Mannosylated (O-Man); this plays an important role in its ability to bind basement membrane proteins containing laminin G domains (Figure 1B).…”

“…Incorporating this architectural design into human Gal‐1 (homo‐oligomer variants ((Gal‐1) 4 ‐GG/8S)) results in a 25‐fold higher cell‐bridging activity than Gal‐1WT, as revealed by hemagglutination and erythrocyte aggregation experiments [12] . Furthermore, a heterotetramer galectin variant with two Gal‐1 and Gal‐3 domains (G1/G3 Zipper) exhibited a higher apoptotic activity than wild‐type Gal‐1 and Gal‐3 alone or in combination; thus, it can be a good therapeutic candidate for regulating innate and adaptive immunity [20,21] …”