A chimeric, multivalent assembly of galectin-1 and galectin-3 with enhanced extracellular activity

Abstract

Assembly of a fusion of galectin-1 and galectin-3 with higher carbohydrate binding affinity and a significantly lower effective dose than galectin-1.

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

“…Intriguingly, the combination of native G1 and G3 was neither additive nor synergistic for activating T cell apoptosis despite engaging different receptors and distinct pathways (Stillman et al, 2006). Consistent with this, we previously reported that a fusion protein consisting of G1 linked to the N-terminus of G3 (i.e., "G1/G3") failed to induce Jurkat T cell apoptosis; however, a synthetic tetramer developed by engineering G1/G3 to self-assemble (i.e., "G1/G3 Zipper") induced apoptosis at a significantly lower concentration than G1 alone (Fettis et al, 2019). We hypothesized that the G1 domains acted as the signaling units, while the G3 domains served as anchors that increase the apparent glycan-binding affinity (Figure 1, left).…”

“…Both caspase-8 and JNK activation can induce changes in metabolic activity that lead to pro-apoptotic signaling (Li et al, 1998;Dhanasekaran and Reddy, 2017). We previously reported that G1/G3 Zipper decreased NADH content of Jurkat T cells to a greater extent than both native G1 and a polymer-stabilized G1 homodimer, as measured by conversion of resazurin to resorufin with the CellTiter-Blue R cell viability assay (Promega) (Fettis et al, 2019). Here we observed that both caspase-8 and JNK activation are involved in pro-apoptotic signaling via G1.…”

“…All proteins in this study were expressed from recombinant DNA in Origami TM B(DE3) Competent E. coli (70837-4, Novagen) and purified according to established protocols (Fettis et al, 2019). Protein sequences of G1, which has been mutated to lack surface cysteines, G3, and G1/G3 Zipper have been published elsewhere (Restuccia et al, 2018;Fettis et al, 2019). After purification, molecular weight and purity of each protein were determined via denaturing gel electrophoresis and Coomassie staining.…”

“…Here, we used a combination of annexin V staining of phosphatidylserine and a membrane-impermeable DNA-binding dye (LIVE/DEAD R , Invitrogen) to quantify Jurkat T cell death induced by G1/G3 Zipper, G1, or G3 as a function of galectin concentration and time. Note, cells were treated with as low as 0.5 µM G1/G3 Zipper in all experiments based on a prior report showing that high concentration of this protein led to loss of cell integrity (Fettis et al, 2019), whereas cells were treated with as high as 5 µM G1 or G3 based on the reported effective dose of these proteins (Restuccia et al, 2015(Restuccia et al, , 2018Farhadi et al, 2018;Fettis and Hudalla, 2018;Fettis et al, 2019). For each concentration of galectin (0.5, 1, 2.5, and 5 µM) tested, G1/G3 Zipper induced significantly more cell death by 24 h than an equimolar combination of G1 and G3 (Figure 2A).…”

Influence of protein (human galectin-3) design on aspects of lectin activity

“…In combination, the reported profiles of staining and glycocluster inhibition when systematically testing the eight proteins with the Gal-3 CRD as common unit give direction to proceed to investigate the architecture-activity relationship in functional assays. Looking at our data on cell-typedependent differences of staining in sections, an extrapolation of functional data for variants obtained in a special cell system, for example for heterodimers and neuroblastoma cells (Ludwig et al 2019b) or for Gal-1/-3 heterotetramers and T leukemic cells (Fettis et al 2019), to any other system appears to be precluded. Fig.…”